MAP kinase modulators and uses thereof

ABSTRACT

The invention provides for novel MAP kinase inhibitors and compositions comprising the same. In some embodiments, the MAP kinase inhibitors are p38α MAP kinase inhibitors. The invention further provides for methods for treatment of diseases comprising administration of MAP kinase inhibitors or compositions comprising MAP kinase inhibitors. In some embodiments, the disease is Alzheimer&#39;s Disease, ALS, Huntington&#39;s Disease or Parkinson&#39;s Disease.

This application is a continuation of U.S. application Ser. No.15/697,656, filed Sep. 7, 2017, which is a divisional of U.S.application Ser. No. 14/855,035, filed Sep. 15, 2015, now U.S. Pat. No.9,783,525, which is a continuation-in-part of International PatentApplication No. PCT/US2014/030260, filed Mar. 17, 2014, which claimspriority to U.S. Provisional Patent Application No. 61/790,207, filedMar. 15, 2013, the entire contents of which are hereby incorporated byreference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant AG043415awarded by the National Institutes of Health. The Government has certainrights in this invention.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

Protein kinases related to mitogen activated protein kinase (MAPK or MAPkinase) are part of stress-related signal transduction pathways. p38MAPK is a serine/threonine kinase that has recently become a target formodulation of cytokine production as it is implicated in multiplesignaling pathways activated during inflammation. Inhibitors of p38 MAPKare being studied for treatment of inflammatory diseases such asrheumatoid arthritis, psoriasis and pain.

Alzheimer's Disease (AD) is a progressive and terminal conditioncharacterized by debilitating memory loss and extensive deterioration ofcognitive and functional abilities. Currently available therapies for ADare palliative and neither cure nor arrest progression of the disease.Cholinesterase inhibitors such as Razadyne® (galantamine), Exelon®(rivastigmine), Aricept® (donepezil), and Cognex® (tacrine) have beenprescribed for early stages of AD, and may temporarily delay or haltprogression of symptoms. However, as AD progresses, the brain loses lessacetylcholine, thereby rendering cholinesterase inhibitors ineffective.Namenda® (memantine), an N-methyl D-aspartate (NMDA) antagonist, is alsoprescribed to treat moderate to severe Alzheimer's disease; however onlytemporary benefits are realized.

There is a need for novel MAP kinase inhibitors. There is also a needfor novel treatments for a variety of disease states for which MAPkinase is implicated. There is a further need for novel and effectivetreatments for neurodegenerative diseases and neurological disorders. Inparticular, there is a continuing need for treatments of dementia andmemory loss associated with Alzheimer's Disease.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a class of compounds offormula (I)

-   wherein-   X₁ is N and X₂ is CH, or X₁ is CH and X₂ is N;-   R¹ is —N(R⁴)₂, cyclopropyl, or R⁵-piperidin-4-yl;-   R² is independently D or halogen;-   R³ is naphthyl, quinolinyl, isoquinolinyl, or indolyl, wherein said    naphthyl, quinolinyl, isoquinolinyl or indolyl is optionally    independently substituted with at least one D, halogen,    (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D;-   R⁴ is independently H, (C₁-C₄)-alkyl, (C₁-C₄)-alkyl substituted with    at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the    nitrogen to which they are attached form a 3-7 membered heterocyclic    ring, wherein one of the carbon atoms is optionally replaced with    NR⁶, O or S, wherein the 3-7 membered heterocyclic ring is    optionally substituted with a (C₁-C₃)-alkyl;-   R⁵ is H or (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least    one D;-   R⁶ is H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with at least one    D, or pyrimidin-2-yl; and-   n is an integer from 0-4; or a pharmaceutically acceptable salt    thereof,-   wherein when R³ is indol-4-yl and n is 0, R¹ is not    N-methyl-piperazinyl.

In another aspect, the invention is directed to compositions comprisinga compound of formula (I) and a pharmaceutically acceptable carrier.

In another aspect, the invention is directed to a method of treatingneurodegeneration in a subject comprising administration of atherapeutically effective amount of a compound of formula (I).

In another aspect, the invention is directed to a method of treatingneurodegenerative disease in a subject comprising administration of atherapeutically effective amount of a compound of formula (I).

In another aspect, the invention is directed to a method of increasinglong-term potentiation in a subject having a neurodegenerative diseasecomprising administration of a therapeutically effective amount of acompound of formula (I).

In another aspect, the invention is directed to a method of improvingmemory in a subject having a neurodegenerative disease comprisingadministration of a therapeutically effective amount of a compound offormula (I).

In another aspect, the invention is directed to a method of improvingsynaptic function in a subject having a neurodegenerative diseasecomprising administration of a therapeutically effective amount of acompound of formula (I).

In another aspect, the invention is directed to a method of restoringmemory loss in a subject having a neurodegenerative disease comprisingadministration of a therapeutically effective amount of a compound offormula (I).

In some embodiments, the disease is ALS, Huntington's Disease,Parkinson's Disease or Alzheimer's Disease. In some embodiments, thedisease is ALS, Parkinson's Disease or Alzheimer's Disease. In someembodiments, the disease is ALS. In some embodiments, the disease isHuntington's Disease. In some embodiments, the disease is Parkinson'sDisease. In some embodiments, the disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of treatingischemia in a subject in need thereof comprising administration of atherapeutically effective amount of a compound of formula (I).

Still other objects and advantages of the invention will become apparentto those of skill in the art from the disclosure herein, which is simplyillustrative and not restrictive. Thus, other embodiments will berecognized by the skilled artisan without departing from the spirit andscope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-F show MW-181 (1) suppresses (A) IL-1β production, (B)phospho-MSK1, (C) phospho-MK2 in BV-2 microglia cell line, (D) IL-1β inprimary microglia from WT mice but not from p38α (T106M) KI microglia,(E) LPS-induced IL-1β increase in mouse cortex of WT mice, (F) IL-1βlevels in cortex of APP/PS1 mice back to control levels after single 2.5mg/kg administration, 3×/week over 5 months.

FIGS. 2A-E show cellular target engagement and mechanism of action ofMW-108 (2). (A). MW-108 inhibited phosphorylation of the p38α substrateMK2 in a concentration-dependent manner in LPS-stimulated BV-2microglial cells. (B). MW-108 inhibited LPS-induced IL-1β production ina concentration-dependent manner in BV-2 cells. (C). MW-108 suppressedIL-1β production in BV-2 cells stimulated with diverse TLR ligands. BV-2cells were treated with ligands for TLR2, TLR4, TLR7/8, and TLR9 in theabsence (white bars) or presence (gray bars) of MW-108. ‡p<0.0001compared to TLR ligand in the absence of compound. (D). MW-108 inhibitedLPS induced IL-1β production in primary microglia from wild-type (WT)mice, but not from drug resistant p38α MAPK knock-in (p38α KI) mice.#p<0.01, § p<0.001 compared to LPS alone. (E). MW-108 suppressedLPS-induced IL-1β levels in vivo. **p<0.01 compared to LPS alone (n=6saline/vehicle; n=24 LPS/vehicle; n=15 LPS/MW-108). Data in all panelsare expressed as a percent of the maximal levels, where levels in thepresence of stressor alone were normalized to 100%.

FIGS. 3A-E show cellular target engagement and mechanism of action ofMW-181 (1). (A). MW-181 inhibited phosphorylation of the p38α substrateMK2 in a concentration-dependent manner in LPS stimulated BV-2microglial cells. (B). MW-181 inhibited LPS-induced IL-1β production ina concentration-dependent manner in BV-2 cells. (C). MW-181 suppressedIL-1β production in BV-2 cells stimulated with diverse TLR ligands. BV-2cells were treated with ligands for TLR2, TLR4, TLR7/8, and TLR9 in theabsence (white bars) or presence (gray bars) of MW-181. ‡p<0.0001compared to TLR ligand in the absence of compound. (D) MW-181 inhibitedLPS-induced IL-1β production in primary microglia from wild-type (WT)mice, but not from drug-resistant p38α MAPK knock-in (p38α KI) mice.#p<0.01, § p<0.001 compared to LPS alone. (E) MW-181 suppressedLPS-induced IL-1β levels in vivo. **p<0.01 compared to LPS alone (n=6saline/vehicle; n=24 LPS/vehicle; n=15 LPS/MW-181). Data in all panelsare expressed as a percent of the maximal levels, where levels in thepresence of stressor alone were normalized to 100%.

FIGS. 4A-C show (A) phosphorylation state of pMK2 is increased at 1 hrafter LPS addition and the increase is attenuated in a dose dependentmanner by MW-181 (1); (B) phosphorylation state of pMSK1 is increased at1 hr after LPS addition and the increase is attenuated in a dosedependent manner by MW-181; and (C) IL-1β protein levels in cell lysatesat 18 hrs after LPS addition in MSD ELISA measurements with vehicle,LPS+vehicle and LPS+MW-181.

FIGS. 5A-B show linkage of in vitro inhibition to in vivo efficacy.MW-181 reduces IL-1β levels (5A) and IL-6 levels (5B) in stressed wildtype (WT) mice but not in drug resistant strains (KI).

FIGS. 6A-C show (A) phosphorylation state of pMK2 is increased at 1 hrafter LPS addition and the increase is attenuated in a dose dependentmanner by MW-150 (27); (B) IL-1β protein levels in cell lysates at 18hrs after LPS addition in MSD ELISA measurements with vehicle,LPS+vehicle and LPS+MW-150; and (C) TNFa protein levels in conditionedmedia at 18 hrs after LPS addition in MSD ELISA measurements withvehicle, LPS+vehicle and LPS+MW-150.

FIGS. 7A-B show two-day RAWM performance is impaired following treatmentwith 200 nM Aβ and in APP/PS1 mice. (A). Mice that received bilateralinjections of Aβ₄₂ [200 nM in a final volume of 1 μl over 1 min; twiceon each testing day: 15 min prior to the 1st trial (for the 1st group oftests) and 15 min prior to the 7th trial (for the 2nd group of tests)])into dorsal hippocampi, failed to reach the learning criterion (˜1error) by block 8 and 9 of day 2 compared to vehicle-infused mice (n=8for both; P<0.001). (B). APP/PS1 mice failed to reach the criterion over2 days of testing compared to WT littermate.

FIGS. 8A-I show beneficial effect of MW-181 (1) on Aβ₄₂-induced synapticand cognitive dysfunction. (A) MW-181 (10 μM) ameliorated the LTPdeficit in Aβ₄₂-treated slices (vehicle: n=7 slices; Aβ: n=8; Aβ+MW-181: n=7; MW-181: n=7; F(1,13)=6.651, p=0.0229, compared to slicestreated only with Aβ₄₂). The horizontal bar represents the time ofapplication of Aβ₄₂ and MW-181. Error bars indicate SEM in this and theremaining panels. (B) MW-181 (5 mg/kg, i.p., 30 min before the 1st and2nd group of tests for the RAWM) ameliorated the reference memorydeficit in Aβ₄₂-infused mice. Aβ₄₂ (200 nM, in a final volume of 1 μlover 1 min) was bilaterally infused 20 min prior to the 1st trial (forthe 1st group of tests in a 2-day RAWM test assessing reference memoryand 20 min prior to the 7th trial (for the 2nd group of tests of theRAWM), into dorsal hippocampus (vehicle: n=10 animals; Aβ: n=10; Aβ+MW-181: n=10; MW-181: n=10; F(1,18)=5.851, p=0.0264 comparing Aβ-infusedmice vs. compound+Aβ infused animals). (C) MW-181 (5 mg/kg, i.p. 30 minbefore the electric shock) ameliorated the contextual fear memorydeficit in Aβ₄₂-infused mice. Aβ₄₂ (200 nM, in a final volume of 1 μlover 1 min) was given 20 min prior to the foot shock (vehicle: n=15animals; Aβ: n=15; Aβ+ MW-181: n=16; MW-181: n=15; p=0.0005 comparingAβ-infused mice vs. compound+Aβ infused animals). (D, E) No differencewas detected between different groups of mice regardless of treatmentwith MW-181 (5 mg/kg, i.p., 30 min before testing) with and without Aβ₄₂(200 nM, in a final volume of 1 μl over 1 min, 20 min before testing)when tested for visual-motor-motivational deficits with the visibleplatform test; speed and time to the platform are shown in D and E,respectively (vehicle: n=10; Aβ: n=10; Aβ+ MW-181: n=10; MW-181: n=10;p>0.05 comparing Aβ-infused mice vs. compound+Aα infused animals forboth). (F) The same animals that underwent contextual fear conditioningtesting were assessed for cued learning 24 hrs after the contextuallearning. No difference was detected between different groups of miceregardless of treatment with MW-181 with and without Aβ₄₂ (vehicle: n=15animals; Aβ: n=15; Aβ+ MW-181: n=16; MW-181: n=15; p>0.05 comparingAβ-infused mice vs. compound+Aα infused animals). (G) Sensory thresholdwas not affected regardless of treatment with MW-108 with and withoutAβ₄₂ (vehicle: n=9 animals; Aβ: n=9; Aβ+ MW-108: n=9; MW-108: n=9;p>0.05 comparing Aβ-infused mice vs. compound+Aβ infused animals). (H,I) No difference in exploratory behavior as shown by a similarpercentage of time spent in the center compartment (H) and the number ofentries into the center compartment (I) were observed regardless oftreatment with MW-181 with and without Aβ₄₂ (vehicle: n=9 animals; Aβ:n=9; Aβ+ MW-181: n=9; MW-181: n=9; p>0.05 comparing Aβ-infused mice vs.compound+Aα infused animals).

FIGS. 9A-I show beneficial effect of MW-108 (2) on Aβ₄₂-induced synapticand cognitive dysfunction. (A) MW-108 (10 μM) ameliorated the LTPdeficit in Aβ₄₂-treated slices (vehicle: n=7 slices; Aβ: n=7; Aβ+MW-108: n=8; MW-108: n=8; F(1,13)=17.25, p=0.0011, compared to slicestreated only with Aβ₄₂). The horizontal bar represents the time ofapplication of Aβ₄₂ and MW-108. Error bars indicate SEM in this and theremaining panels. (B) MW-108 (5 mg/kg, i.p., 30 min before the 1st and2nd group of tests for the RAWM) ameliorated the reference memorydeficit in Aβ₄₂-infused mice. Aβ₄₂ (200 nM, in a final volume of 1 μlover 1 min) was bilaterally infused 20 min prior to the 1st trial (forthe 1st group of tests in a 2-day RAWM test assessing reference memoryand 20 min prior to the 7th trial (for the 2nd group of tests of theRAWM), into dorsal hippocampus (vehicle: n=10 animals; Aβ: n=12; Aβ+MW-108: n=14; MW-108: n=13; F(1,24)=1.827, p=0.0001 comparing Aβ-infusedmice vs. compound+Aβ infused animals). (C) MW-108 (5 mg/kg, i.p. 30 minbefore the electric shock) ameliorated the contextual fear memorydeficit in Aβ₄₂-infused mice. Aβ₄₂ (200 nM, in a final volume of 1 μlover 1 min) was given 20 min prior to the foot shock (vehicle: n=16animals; Aβ: n=17; Aβ+ MW-108: n=17; MW-108: n=18; p=0.009 comparingAβ-infused mice vs. compound+Aβ infused animals). (D-E) No differencewas detected between different groups of mice regardless of treatmentwith MW-108 (5 mg/kg, i.p., 30 min before testing) with and without Aβ₄₂(200 nM, in a final volume of 1 μl over 1 min, 20 min before testing)when tested for visual-motor-motivational deficits with the visibleplatform test; speed and time to the platform are shown in D and E,respectively (vehicle: n=10; Aβ: n=12; Aβ+ MW-108: n=14; MW-108: n=13;p>0.05 comparing Aβ-infused mice vs. compound+Aβ infused animals forboth). (F) The same animals that underwent contextual fear conditioningtesting were assessed for cued learning 24 hrs after the contextuallearning. No difference was detected between different groups of miceregardless of treatment with MW-108 with and without Aβ₄₂ (vehicle: n=16animals; Aβ: n=17; Aβ+ MW-108: n=17; MW-108: n=18; p>0.05 comparingAβ-infused mice vs. compound+Aβ infused animals). (G) Sensory thresholdwas not affected regardless of treatment with MW-108 with and withoutAβ₄₂ (vehicle: n=13 animals; Aβ: n=16; Aβ+ MW-108: n=17; MW-108: n=15;p>0.05 comparing Aβ-infused mice vs. compound+Aβ infused animals). (H-I)No difference in exploratory behavior as shown by a similar percentageof time spent in the center compartment (H) and the number of entriesinto the center compartment (I) were observed regardless of treatmentwith MW-108 with and without Aβ₄₂ (vehicle: n=13 animals; Aβ: n=16; Aβ+MW-108: n=17; MW-108: n=15; p>0.05 comparing Aβ-infused mice vs.compound+Aβ infused animals).

FIGS. 10A-C show (A) potentiation as a percent of baseline in WT,APP/PS1, WT+MW-181 (1), and APP/PS1+MW-181 treated mice; (B) RAWMresults for WT, APP/PS1, WT+MW-181, and APP/PS1+MW-181 treated mice; and(C) FC results for WT, APP/PS1, WT+MW-181, and APP/PS1+MW-181 treatedmice. MW-181 is beneficial against the defects in LPT (A), RAWMperformance (B) and FC (C).

FIGS. 11A-J show (A) potentiation as a percent of baseline in WT,APP/PS1, WT+MW-108 (2), and APP/PS1+MW-108 treated mice; (B) RAWMresults for WT, APP/PS1, WT+MW-108, and APP/PS1+MW-108 treated mice; and(C) FC results for WT, APP/PS1, WT+MW-108, and APP/PS1+MW-108 treatedmice. The remaining panels show controls for input/output relationship(D), latency and speed to the visible platform (E, F), cued conditioning(G), sensory threshold assessment (H), number of entries into the center(I) and percent time spent in the center (J) during open fieldassessment.

FIGS. 12A-C show contextual fear conditioning results for (A)WT+vehicle, APP/PS1+vehicle, WT+MW-077 (7), and APP/PS1+MW-077 treatedmice (WT+vehicle: n=14; APP/PS1+vehicle: n=13; WT+MW-077: n=13;APP/PS1+MW-077: n=12). At 24 hours, statistical analyses showed: APP/PS1vehicle vs. APP/PS1+MW-077: p=0.00006099; APP/PS1 vehicle vs.WT-vehicle: p=0.00000094; (B) WT+vehicle, APP/PS1+vehicle, WT+MW-125(9), and APP/PS1+MW-125 treated mice (WT+vehicle: n=14; APP/PS1+vehicle:n=13; WT+MW-125: n=13; APP/PS1+MW-125: n=12). At 24 hours, statisticalanalyses showed: APP/PS1 vehicle vs. APP/PS1+MW-125: p=0.00224081;APP/PS1 vehicle vs. WT-vehicle: p=0.00000094; and (C) WT+vehicle,APP/PS1+vehicle, WT+MW-150 (27), and APP/PS1+MW-150 treated mice(WT+vehicle: n=10; APP/PS1+vehicle: n=10; WT+MW-150: n=11;APP/PS1+MW-150: n=10). At 24 hours, statistical analyses showed: APP/PS1vehicle vs. APP/PS1+MW-125: p=0.00148599; APP/PS1 vehicle vs.WT-vehicle: p=0.00032711.

FIGS. 13A-C show cued fear conditioning results for (A) WT+vehicle,APP/PS1+vehicle, WT+MW-077(7), and APP/PS1+MW-077 treated mice(WT+vehicle: n=14; APP/PS1+vehicle: n=13; WT+MW-077: n=13;APP/PS1+MW-077: n=12); (B) WT+vehicle, APP/PS1+vehicle, WT+MW-125 (9),and APP/PS1+MW-125 treated mice (WT+vehicle: n=14; APP/PS1+vehicle:n=13; WT+MW-125: n=13; APP/PS1+MW-125: n=12); and (C) WT+vehicle,APP/PS1+vehicle, WT+MW-150 (27), and APP/PS1+MW-150 treated mice(WT+vehicle: n=10; APP/PS1+vehicle: n=10; WT+MW-150: n=11;APP/PS1+MW-150: n=10).

FIGS. 14A-B show open field results for (A) time spent in the center,and (B) number of entries into center for WT+vehicle, APP/PS1+vehicle,WT+MW-077 (7), and APP/PS1+MW-077 treated mice (WT+vehicle: n=14;APP/PS1+vehicle: n=13; WT+MW-077: n=13; APP/PS1+MW-077: n=12).

FIGS. 15A-B show open field results for (A) time spent in the center,and (B) number of entries into center for WT+vehicle, APP/PS1+vehicle,WT+MW-125 (9), and APP/PS1+MW-125 treated mice (WT+vehicle: n=14;APP/PS1+vehicle: n=13; WT+MW-125: n=13; APP/PS1+MW-125: n=12).

FIGS. 16A-B show open field results for (A) time spent in the center,and (B) number of entries into center for WT+vehicle, APP/PS1+vehicle,WT+MW-150 (27), and APP/PS1+MW-150 treated mice (WT+vehicle: n=10;APP/PS1+vehicle: n=10; WT+MW-150: n=11; APP/PS1+MW-150: n=10).

FIG. 17 shows sensory threshold assessment results for WT+vehicle,APP/PS1vehicle, WT+MW-077 (7), and APP/PS1+MW-077 treated mice (WT:n=14; APP/PS1: n=13; WT+MW-077: n=13; APP/PS1+MW-077: n=12).

FIG. 18 shows sensory threshold assessment results for WT+vehicle,APP/PS1+vehicle, WT+MW-125 (9), and APP/PS1+MW-125 treated mice(WT+vehicle: n=14; APP/PS1+vehicle: n=13; WT+MW-125: n=13;APP/PS1+MW-125: n=12).

FIG. 19 shows sensory threshold assessment results for WT+vehicle,APP/PS1+vehicle, WT+MW-150 (27), and APP/PS1+MW-150 treated mice(WT+vehicle: n=10; APP/PS1+vehicle: n=10; WT+MW-150: n=11;APP/PS1+MW-150: n=10).

FIGS. 20A-C show time to platform results in visible platform test in(A) WT+vehicle, APP/PS1+vehicle, WT+MW-077 (7), and AP/PS1+MW-077treated mice (WT+vehicle: n=14; APP/PS1+vehicle: n=13; WT+MW-077: n=13;APP/PS1+MW-077: n=12); (B) WT+vehicle, APP/PS1+vehicle, WT+MW-125 (9),and AP/PS1+MW-125 treated mice (WT+vehicle: n=14; APP/PS1+vehicle: n=13;WT+MW-125: n=13; APP/PS1+MW-125: n=12); and (C) WT+vehicle,APP/PS1+vehicle, WT+MW-150 (27), and AP/PS1+MW-150 treated mice(WT+vehicle: n=10; APP/PS1+vehicle: n=10; WT+MW-150: n=11;APP/PS1+MW-150: n=10).

FIGS. 21A-C show speed to platform results in visible platform test in(A) WT+vehicle, APP/PS1+vehicle, WT+MW-077 (7), and AP/PS1+MW-077treated mice (WT+vehicle: n=14; APP/PS1+vehicle: n=13; WT+MW-077: n=13;APP/PS1+MW-077: n=12): (B) WT+vehicle, APP/PS1+vehicle, WT+MW-125 (9),and AP/PS1+MW-125 treated mice (WT+vehicle: n=14; APP/PS1+vehicle: n=13;WT+MW-125: n=13; APP/PS1+MW-125: n=12): and (C) WT+vehicle,APP/PS1+vehicle, WT+MW-150 (27), and AP/PS1+MW-150 treated mice(WT+vehicle: n=10; APP/PS1+vehicle: n=10; WT+MW-150: n=11;APP/PS1+MW-150: n=10).

FIGS. 22A-C show RAWM results in (A) WT+vehicle, APP/PS1+vehicle,WT+MW-077 (7), and AP/PS1+MW-077 treated mice (WT+vehicle: n=14;APP/PS1+vehicle: n=14; WT+MW-077: n=13; APP/PS1+MW-077: n=12).Statistical analyses showed: APP/PS1 vehicle vs. APP/PS1+MW-077:p=0.14056485 (3^(rd) last point), 0.00166180 (2^(nd) last point),0.00039962 (last point); APP/PS1 vehicle vs. WT-vehicle: p=0.00354140(3^(rd) last point), 0.00007920 (2^(nd) last point), 0.00000630 (lastpoint): (B) WT+vehicle, APP/PS1+vehicle, WT+MW-125 (9), andAP/PS1+MW-125 treated mice (WT+vehicle: n=14; APP/PS1+vehicle: n=14;WT+MW-125: n=13; APP/PS1+MW-125: n=12). Statistical analyses showed:APP/PS1 vehicle vs. APP/PS1+MW-125: p=0.01969560 (3^(rd) last point),0.00269137 (2^(nd) last point), 0.00079845 (last point); APP/PS1 vehiclevs. WT-vehicle: p=0.00354140 (3^(rd) last point), 0.00007920 (2^(nd)last point), 0.00000630 (last point); and (C) WT+vehicle,APP/PS1+vehicle, WT+MW-150 (27), and AP/PS1+MW-150 treated mice(WT+vehicle: n=10; APP/PS1+vehicle: n=10; WT+MW-150: n=11;APP/PS1+MW-150: n=10). Statistical analyses showed: APP/PS1 vehicle vs.APP/PS1+MW-150: p=0.04494522 (3^(rd) last point), 0.12684352 (2^(nd)last point), 0.02070092 (last point); APP/PS1 vehicle vs. WT-vehicle:p=0.01189036 (3^(rd) last point), 0.04848323 (2^(nd) last point),0.00119455 (last point).

FIGS. 23A-C show potentiation as a percent of baseline in (A)APP/PS1+vehicle (n=12), APP/PS1+MW-077 (7) (n=10), WT+vehicle (n=14),WT+MW-077 (n=14). APP/PS1 vehicle vs. APP/PS1+MW-077: Two-way ANOVAF(1,19)=5.163, p=0.0349. APP/PS1 vehicle vs. WT+vehicle: F(1,24)=5.967,p=0.0223; (B) APP/PS1+vehicle (n=12), APP/PS1+MW-125 (9) (n=9),WT+vehicle (n=14), WT+MW-125 (n=7). APP/PS1 vehicle vs. APP/PS1+MW-077:Two-way ANOVA F(1,19)=9.357 p=0.0065. APP/PS1 vehicle vs. WT+vehicle:F(1,24)=5.967, p=0.0223; and (C) APP/PS1+vehicle (n=12), APP/PS1+MW-150(27) (n=7), WT+vehicle (n=14), and WT+MW-150 (n=9). APP/PS1 vehicle vs.APP/PS1+MW-077: Two-way ANOVA F(1,17)=6.780 P=0.0185. APP/PS1 vehiclevs. WT+vehicle: F(1,24)=5.967, p=0.0223.

FIG. 24 shows synaptic depression induced by oxygen glucose deprivation(OGD) is ameliorated by MW-150 (27). After 15 min of stable baseline,oxygen glucose deprivation was applied for 10 min (dark bar).

FIGS. 25A-25B show effect of MW-150 treatment for behavior end points.Dose response curve for MW-150 during assessment of associative andspatial memory deficit in APP/PS1 transgenic mice. Mice wereadministered orally either saline vehicle or different concentrations ofMW-150 daily from 8 weeks of age until 3-4 months when associative andspatial memories were assessed through contextual fear memory (FIG. 25A)and RAWM (FIG. 25B). RAWM errors correspond to the number of errors thatmice (3-15 per test) made at the last set of trials.

FIGS. 26A-26D show effect of MW-150 treatment on cognitive performancein the absence of effect on A-β plaque burden. Sections of cortex fromAPP/PS1 transgenic mice treated with vehicle (FIG. 26A) or MW-150 (FIG.26B) were stained with 6E10 anti-Aβ antibody (10× objective; 100×magnification). (FIG. 26C) Quantification by a board-certifiedneuropathologist blinded to the treatment groups was done by analysis ofa brain sagittal section from each mouse in which the total number ofwell-formed β-amyloid plaques in the entire section were counted. Errorbars show standard error of the mean (n=4 for each group). (FIG. 26D) Ina separate experiment with APP/PS1 knock-in (KI) mice, Aβ plaques werequantified from KI mice treated with MW-150 or vehicle. In both AD mouseS15 models, there are no effects of MW-150 treatment on the amyloidplaque burden.

DETAILED DESCRIPTION OF THE INVENTION

Glia are innate immunity cells of the central nervous system (CNS) thatare key to homeostasis and health. However, they can be transformed intoactivated or effector type cells capable of releasing small proteinssuch as cytokines and other cytoactive factors that contribute tocomplex disease states. In some cases, transformed glia can becomeautonomous by creating a microenvironment, such as in glioma, wherethere is increased production of proinflammatory cytokines anddysfunction in signaling pathways. Up-regulated cytokine production byother types of transformed glia can result in dysfunction in a criticalphysiological axis, such as the perturbation of glia-neuronalinteractions. This can result in synaptic dysfunction, a common elementof disease progression evident in diverse neurodegenerative diseases,post traumatic injury or complex syndromes evident in aged individuals.The term “synaptopathy” is often used to encompass a series of diseasesthat, despite manifesting with different symptoms, have much more incommon that clinically evident. The need for neurons to maintain healthyand functional synapses is challenging as high polarization with axons,branched dentritic trees and numerous synaptic contacts is a keyfeature. A consequence of faltering synapses is onset of numerousneurodegenerative disorders, many with aging. Molecular mechanismsrelevant to normal synaptic function are altered in the states, leadingto synaptopathy, often in a cause/effect relationship that is connectedwith etiopathogenesis of the specific disease, or as a side consequenceof the alteration of the mechanisms underlying synaptic transmission.

A variety of efforts are being expended to control the physiologicaleffects of glia transformation as a sequealae of acute injury and as onecomponent of progressive diseases such as neurodegeneration, dementiaand glioma proliferation/metastasis. A prevailing theme is a focus onmodulation of intracellular pathways in glia and their linkage tointercellular or organismal networks. The intracellular stress activatedpathways controlled by enzymes called protein kinases are often targetedwith small molecule drugs that inhibit multiple protein kinases inparallel or by targeting a single kinase with a causative linkage todisease or pathology progression. One of the single molecular targetefforts are those focused on the stress activated kinase calledp38·MAPK, a regulator of cellular stress response and phenotypicswitching.

Protein kinase inhibitors for CNS disorders are preferably CNS penetrantand avoid inhibition of other key signaling pathways that may contributeto adverse events. There is interest in novel small molecules fordisease modification where glia functions are involved in the pathologyprogression.

p38 MAPK, a stress related protein kinase involved in eukaryotic signaltransduction, has recently become the focus of drug discovery effortstargeted at neurodegeneration and central nervous system relateddiseases. p38 MAPK is a serine/threonine kinase, which exists as atleast four isoforms or subtypes, namely α, β (and P2), δ, and γ, each ofwhich are encoded by separate genes and have varying sensitivity toinhibitors.

Intracellular protein kinases such as p38 MAPK, sometimes called stresskinases due to their activation with injury and disease, activate acascade of downstream events in the cell that can result in pathologywithin a given tissue. When there is coincident activation of thesepathways in integrated cellular networks, such as glia and neurons inthe central nervous system, the impact of the system can be significantdue to potential synergies among cells in the network. In the CNS, thiscan be manifest in cellular dysfunctions such as intracellular fastaxonal transport or intercellular connections such as synapses. Theseeffects are due to internal changes within neurons, with the biologicaleffects potentially amplified through the activation of the p38 MAPKmediated pathways within glia, which can result in overproduction ofproinflammatory cytokines that further stress or injure the neurons.Therefore, the CNS offers an interesting opportunity to target the samemolecular target in the two distinct major cell types, glia and neurons,in order to modulate the system wide pathology via attenuation ofintracellular cascades regulated by p38 MAPK. Synaptic dysfunction, inwhich activation of p38 MAPK has been established as a causativecontributor, is considered a fundamental event in diverse diseases andinjury sequelae, including, for example, Alzheimer's disease, ALS,dementias, cognitive impairment and morbidity from injury, and otherneurological diseases. Therefore, the availability of highly selective,CNS penetrant, p38 MAPK inhibitor drugs would be a major contributiontowards developing new therapeutic paradigms for disease alteration.Potential applications further include therapeutic intervention for CNSdiseases and injuries, oncology where tumor immunology andmicroenvironment involves cytokine alterations that are regulated by p38MAPK, and other applications related to p38 MAPK.

The alpha isoform, p38a, has been the most thoroughly studied, and is apotential target for modulation of cytokine production as it isimplicated in multiple signaling pathways activated during inflammation(Dominguez C. et al., Curr. Opin. Drug Disc. Dev. 2005, 8, 421-430;herein incorporated by reference in its entirety). Indeed, p38 MAPKinhibitors are in clinical trials to treat inflammatory diseases such asrheumatoid arthritis, psoriasis and pain (“Competitor Analysis-p38 MAPKinhibitors 2010” La Merie Publishing, p 21, 2010(http://www.researchandmarkets.com/research/bee137/competitor_analysi);herein incorporated by reference in its entirety). There is alsoaccumulating evidence that p38 MAPK plays a role in AD. AD ischaracterized by neuroinflammation due to glial cell activation and theproduction of inflammatory mediators (Munoz, L. and Ammit, A.,Neuropharmacology, 2010, 58, 561-568; Munoz, L., et al., J.Neuroinflammation, 2007, 4, 21; each herein incorporated by reference inits entirety). This inflammation has a detrimental result on ADpathology. AD patients may benefit from treatment with p38 MAPKinhibitors (See, e.g., Watterson, D. M., et al., PLoS One (8)6: e66226(2013); Munoz, L., et al., J. Neuroinflammation, 2007, 4, 21; eachherein incorporated by reference in its entirety). Alzheimer's Disease(AD) is a leading cause of death in the United States, with only fewtreatment options for the afflicted patient. Current treatment optionsare merely palliative and do not cure or arrest progression of thedisease.

AD is not the only disease in which p38 MAPK has been found to bealtered. Animal model studies are consistent with the involvement of thep38α MAPK in AD related pathology progression and in other diseasescharacterized by glia transformation (e.g, ALS and glioma). p38 MAPK isat the crossroad of multiple signaling mechanisms including a) thoseactivated by inflammatory cytokines binding to specific receptors at thecell surface, and promoting activation of interleukin-1 receptorassociated kinase, TNF receptor-associated factor 2/6 leading toactivation of MKKKs, and subsequently phosphorylation of MKK3 and MKK6,that, in turn, activate p38 MAPK; b) those activated by glutamate that,once released from the presynaptic terminal, binds to glutamatereceptors (AMPA and NMDA, as well as metabotropic receptors) and eitherdirectly or indirectly (see metabotropic receptor that activates RAP1which, in turn, phosphorylates MKK3/6) activate p38 MAPK (See, e.g.,Correa et al, J. Signal Transduction 2012, Article ID 649079,doi:10.1155/2012/649079; herein incorporated by reference in itsentirety). Thus, p38 MAPK is involved in a multitude of physiologic andpathologic chains of reactions that eventually lead to normal synapticplasticity, the cellular bases of learning and memory, andneurodegenerative diseases characterized by cognitive disorders andinflammation. Inhibitors of p38 MAPK can be beneficial, for example, inseveral disorders and neurodegenerative diseases characterized byaltered cellular function, synaptic dysfunction, fast axonal transport,and neuroinflammation. Alzheimer's disease, Parkinson's disease,Huntington disease, Down syndrome, head trauma, traumatic brain injury(TBI), brain injury due to cerebral focal ischemia, attention deficitdisorder, neuronal degeneration with brain iron accumulation type I(Hallervorden-Spatz disease), Lytico-Bodig disease (Parkinson-dementiacomplex of Guam), pure autonomic failure, REM sleep behavior disorder,mild cognitive impairment (MCI), cerebral amyloid angiopathy (CAA), mildcognitive deficits, aging, vascular dementias mixed with Alzheimer'sdisease, any neurodegenerative disease characterized by abnormal amyloiddeposition, or any combination thereof, Adrenoleukodystrophy (ALD),Alcoholism, Alexander's disease, Alper's disease, Amyotrophic lateralsclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease(also known as Spielmeyer-Vogt-Sjögren-Batten disease), Bovinespongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome,Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatalinsomnia, Frontotemporal lobar degeneration, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy bodydementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellarataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy,Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Rett's syndrome, Tau-positive FrontoTemporaldementia, Tau-negative FrontoTemporal dementia, Dementia pugilistica(chronic traumatic encephalopathy), Refsum's disease, Sandhoff disease,Schilder's disease, Subacute combined degeneration of spinal cordsecondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease(also known as Batten disease), Spinocerebellar ataxia (multiple typeswith varying characteristics), Spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, or Toxicencephalopathy, Inclusion body myositis. Neuroinflammation has been alsofound to play a role in psychiatric illnesses. Indeed, a link betweenpsychiatric symptoms and autoimmunity in autoimmune diseases,neuroimmunological abnormalities occur in classical psychiatricdisorders (for example, major depressive, bipolar, schizophrenia, andobsessive compulsive disorders).

Proof of concept studies show that the p38 MAPK inhibitors rescue theAlzheimer's disease phenotype in animal models. These types ofinhibitors can be used against brain injury and neuronal deficits incerebral focal ischemia (Barone et al, J. of Pharmacology and

Experimental Therapeutics. 296: 312, 2001; herein incorporated byreference in its entirety), as well as to counteract increase ofcytokines. Additionally, increase in activation of p38 MAPK has beenobserved in specific pathologic conditions such as familial forms of ALS(Morfini et al, Plos One 8: e65235, 2013; herein incorporated byreference in its entirety), and therefore p38 MAPK inhibitors can beused in these diseases. Similar to soluble forms of beta amyloid thathave been tested with p38 MAPK inhibitors in the context of Alzheimer'sDisease, and soluble forms of tau have been associated with synapticdysfunction and memory loss. Effect of the inhibitors onto tauopathiessuch as FrontoTemporal dementia, Niemann Pick disease, ProgressiveSupranuclear Palsy, TBI, Dementia pugilistica (chronic traumaticencephalopathy), Lytico-Bodig disease (Parkinsondementia complex ofGuam), lead encephalopathy, tuberous sclerosis, Hallervorden-Spatzdisease, and lipofuscinosis, corticobasal degeneration, Argyrophilicgrain disease (AGD), and Frontotemporal lobar degeneration is also beingstudied. For example, a model of tauopathy has been developed in whichtau oligomers are exogenously administered to the mouse brain, leadingto impairment of synaptic plasticity and memory (Puzzo et al.,Behavioral assays with mouse models of Alzheimer's disease: Practicalconsiderations and guidelines. Biochem Pharmacol. 2014 Jan. 21. pii:S0006-2952(14)00035-5. doi: 0.1016/j.bcp.2014.01.011. [Epub ahead ofprint] Review. PMID: 24462904; herein incorporated by reference in itsentirety).

Thus, testing of compounds of the invention in AD models such as theAPP/PS1 mouse (a transgenic mouse that produces high amounts of amyloidbeta and deposits it in amyloid plaques in the brain) show rescue bothsynaptic plasticity defect and abnormal cognitive function. Consistentwith broader indications for CNS diseases and injuries in which p38 MAPKactivity is increased, compounds of the invention exhibit concentrationdependent suppression of LPS induced proinflammatory cytokine by glia ina peripheral LPS stress test.

Other relevant applications related to MAPK and pharmacological studiesare described, for example, in the following publications, each of whichis incorporated herein by reference in its entirety:

-   Alam J J, Compositions and method for treating Alzheimer's Disease,    WO 2012/154814;-   Alamed J. et al., (2006) Two-day radial-arm water maze learning and    memory task; robust resolution of amyloid-related memory deficits in    transgenic mice. Nat Protoc 1: 1671-1679;-   Bachstetter A D and Van Eldik, L J (2010) The p38 MAP kinase family    as regulators of proinflammatory cytokine production in degenerative    diseases of the CNS. Aging and Disease 1: 199-211;-   Barone F C et al., (2001) SB 239063, a second-generation p38    mitogen-activated protein kinase inhibitor, reduces brain injury and    neurological deficits in cerebral focal ischemia, J. Pharm. Exp.    Ther. 296:312-321;-   Correa S A L, Eales K L (2012) The role of p38 MAPK and its    substrates in neuronal plasticity and neurodegenerative disease, J.    Signal Transduction Article ID 649079;-   Chico L K et al., (2009) Targeting protein kinases in central    nervous system disorders. Nature Rev Drug Discovery 8: 892-909;-   Chico L K et al., (2009) Molecular properties and CYP2D6 substrates:    central nervous system therapeutics case study and pattern analysis    of a substrate database. Drug Metab Disp 37:2204-2211;-   Giovannini, M G et al., (2002) Beta-amyloid-induced inflammation and    cholinergic hypofunction in the rat brain in vivo: involvement of    the p38MAPK pathway. Neurobiol Dis 11: 257-274;-   Glover L E et al. A step-up approach for cell therapy in stroke:    translational hurdles of bone marrow-derived stem cells. Transl    Stroke Res 2012; 3:90-98. PMCID: PMC3284662;-   Gong B et al., (2004) Persistent improvement in synaptic and    cognitive functions in an Alzheimer mouse model after rolipram    treatment. J Clin Invest 114: 1624-1634;-   Graziosi L et al., (2012) Mechanistic role of p38 MAPK in gastric    cancer dissemination in a rodent model peritoneal metastasis,    Eur. J. Pharm. 674:143-152;-   Hensley K et al. (1999) p38 kinase is activated in the Alzheimer's    disease brain. J Neurochem 72: 2053-2058;-   Li Y et al., (2003) Interleukin-1 mediates pathological effects of    microglia on tau phosphorylation and on synaptophysin synthesis in    cortical neurons through a p38-MAPK pathway. J Neurosci 23:    1605-1611;-   Masliah E (1995) Mechanisms of synaptic dysfunction in Alzheimer's    disease. Histol Histopathol 10: 509-519;-   Morfini G A et al., Inhibition of Fast Axonal Transport by    Pathogenic SOD1 Involves Activation of p38 MAP Kinase. PLoS ONE    2013, 8(6): e65235;-   Munoz L et al., (2007) A novel p38 alpha MAPK inhibitor suppresses    brain proinflammatory cytokine up-regulation and attenuates synaptic    dysfunction and behavioral deficits in an Alzheimer's disease mouse    model. J Neuroinflammation 4:21;-   Oddo S et al., (2003) Triple-transgenic model of Alzheimer's disease    with plaques and tangles: intracellular Abeta and synaptic    dysfunction. Neuron 39: 409-421;-   Rowan M J et al., (2003) Synaptic plasticity in animal models of    early Alzheimer's disease. Philos Trans R Soc Lond B Biol Sci 358:    821-828;-   Selkoe D J (2002) Alzheimer's disease is a synaptic failure. Science    298: 789-791;-   Selkoe D J (2008) Soluble oligomers of the amyloid beta-protein    impair synaptic plasticity and behavior. Behav Brain Res 192:    106-113;-   Sheng J G et al., (2001) Interleukin-1 promotion of MAPK-p38    overexpression in experimental animals and in Alzheimer's disease:    potential significance for tau protein phosphorylation. Neurochem    Intl 39: 341-348;-   Sun A et al., (2003) p38 MAP kinase is activated at early stages in    Alzheimer's disease brain. Exp Neurol 183: 394-405;-   Teich A F and Arancio O (2012) Is the Amyloid Hypothesis of    Alzheimer's Disease Therapeutically Relevant? Biochem J. 446:    165-177;-   Tong L et al., (2012) J. Neurosci. 32(49): 17714-17724;-   Vitolo O V et al., Pathway and long-term potentiation: reversibility    by drugs that enhance cAMP signaling. Proc Natl Acad Sci USA 99:    13217-13221;-   Wagner E F and Nebreda A R (2009) Signal integration by JNK and p38    MAPK pathways in cancer development, Nat. Rev. Cancer 9:537-549;-   Watterson D M et al., (2013) Development of novel in vivo chemical    probes to address CNS protein kinase involvement in synaptic    dysfunction. PLOS One 8: e66226;-   Xing B et al., (2011) Microglia p38α MAPK is critical for    LPS-induced neuron degeneration through a mechanism involving TNFα.    Molecular Neurodegeneration 6: 84;-   Walsh D M et al., (2002) Naturally secreted oligomers of amyloid    beta protein potently inhibit hippocampal long-term potentiation in    vivo. Nature 416: 535-539;-   Yang G et al., (2013) Mitogen-activated protein kinases regulate    vascular reactivity after hemorrhagic shock through myosin light    chain phosphorylation pathway, J. Trauma Acute Care Surg.    74:1033-1043; and-   Yeung Y T et al., (2013) Interleukins in glioblastoma    pathophysiology: implications for therapy, Br. J. Pharmacol. 168:    591-606.

MAPK inhibitors are described, for example, in U.S. Pat. Nos. 7,919,485;8,188,096; U.S. Patent Publication Nos. 2010/0104536, 2012/0289511;European Patent No. EP1196167, EP2426134; EP1606283; Kumar, S., et al.,Nat. Rev. Drug Disc. 2003, 2, 717; Munoz, L., et al., J.Neuroinflammation 2007, 4, 21; and Watterson, D. M., et al., PLoS One(8)6: e66226 (2013); and references therein; each herein incorporated byreference in its entirety. Most p38 MAPK inhibitors are multi-kinaseinhibitors and exhibit relatively unselective inhibition of severalkinases such as p38, JNK, ERK and upstream and downstream proteinkinases. Pharmacological profiles of such compounds risk unexpectedoutcomes in terms of efficacy, toxicity and side effect profile. Targetengagement studies were performed along with signal transduction in cellculture and in vivo pharmacology. In some embodiments, the compounds ofthe invention selectively inhibit p38α MAPK. In some embodiments, thecompound of formula (I) is a pyridazine derivative. In some embodiments,the compound of formula (I) is a pyrazine derivative.

In some embodiments of formula (I), X₁ is N and X₂ is CH, or X₁ is CHand X₂ is N;

-   R¹ is —N(R⁴)₂, cyclopropyl, or R⁵-piperidin-4-yl;-   R² is independently D or halogen;-   R³ is naphthyl, quinolinyl, isoquinolinyl, or indolyl, wherein said    naphthyl, quinolinyl, isoquinolinyl or indolyl is optionally    independently substituted with at least one D, halogen,    (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D;-   R⁴ is independently H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with    at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the    nitrogen to which they are attached form a 3-7 membered heterocyclic    ring, wherein one of the carbon atoms is optionally replaced with    NR⁶, O or S;-   R⁵ is H or (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least    one D;-   R⁶ is H, (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least    one D; and-   n is an integer from 0-4; or a pharmaceutically acceptable salt    thereof,-   wherein when R³ is indol-4-yl and n is 0, R¹ is not    N-methyl-piperazinyl.

In some embodiments of formula (I), X₁ is N and X₂ is CH;

-   R¹ is —N(R⁴)₂, cyclopropyl, or R⁵-piperidin-4-yl;-   R² is independently D or halogen;

R³ is naphthyl, quinolinyl, isoquinolinyl, or indolyl, wherein saidnaphthyl, quinolinyl, isoquinolinyl or indolyl is optionallyindependently substituted with at least one D, halogen, (C₁-C₃)-alkoxy,or (C₁-C₃)-alkoxy substituted with at least one D;

-   R⁴ is independently H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with    at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the    nitrogen to which they are attached form a 3-7 membered heterocyclic    ring, wherein one of the carbon atoms is optionally replaced with    NR⁶, O or S;-   R⁵ is H or (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least    one D;-   R⁶ is H, (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least    one D; and-   n is an integer from 0-4; or a pharmaceutically acceptable salt    thereof.

In some embodiments of formula (I), X₁ is CH and X₂ is N;

-   R¹ is —N(R⁴)₂, cyclopropyl, or R⁵-piperidin-4-yl;-   R² is independently D or halogen;-   R³ is naphthyl, quinolinyl, isoquinolinyl, or indolyl, wherein said    naphthyl, quinolinyl, isoquinolinyl or indolyl is optionally    independently substituted with at least one D, halogen,    (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D;-   R⁴ is independently H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with    at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the    nitrogen to which they are attached form a 3-7 membered heterocyclic    ring, wherein one of the carbon atoms is optionally replaced with    NR⁶, O or S;-   R⁵ is H or (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least    one D;-   R⁶ is H, (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least    one D; and-   n is an integer from 0-4; or a pharmaceutically acceptable salt    thereof.

In some embodiments of formula (I), R² is independently halogen;

-   R³ is naphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl,    isoquinolin-5-yl, or indol-5-yl, wherein said naphthalen-1-yl,    naphthalen-2-yl, quinolin-8-yl, isoquinolin-5-yl, or indol-5-yl is    optionally independently substituted with at least one halogen or    (C₁-C₃)-alkoxy;-   R⁴ is independently H, (C₁-C₃)-alkyl, (C₃-C₅)-cycloalkyl, or each R⁴    together with the nitrogen to which they are attached form a 3-7    membered heterocyclic ring, wherein one of the carbon atoms is    optionally replaced with NR⁶, O or S;-   R⁵ is H or (C₁-C₃)-alkyl; and-   R⁶ is H or (C₁-C₃)-alkyl.

In some embodiments of formula (I), R³ is naphthalen-1-yl,naphthalen-2-yl, quinolin-8-yl, or isoquinolin-5-yl, whereinnaphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, or isoquinolin-5-yl isoptionally independently substituted with at least one halogen or(C₁-C₃)-alkoxy;

-   R⁴ is independently H, (C₁-C₃)-alkyl, (C₃-C₅)-cycloalkyl, or each R⁴    together with the nitrogen to which they are attached form N-methyl    piperazine, piperazine, piperidine, pyrrolidine, azetidine, or    morpholine; and-   n is an integer from 0-2.

In some embodiments of formula (I), R¹ is —N(R⁴)2 or cyclopropyl;

-   R² is independently halogen;-   R³ is naphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, or    isoquinolin-5-yl, wherein naphthalen-1-yl, naphthalen-2-yl,    quinolin-8-yl, or isoquinolin-5-yl is optionally independently    substituted with at least one halogen or (C₁-C₃)-alkoxy;

R⁴ is independently H, (C₁-C₃)-alkyl, (C₃-C₅)-cycloalkyl, or each R⁴together with the nitrogen to which they are attached formNR⁶-piperazine, piperidine, pyrrolidine, azetidine, or morpholine;

-   R⁶ is H, methyl or CD₃; and-   and n is an integer from 0-2.

In some embodiments of formula (I), R¹ is —N(R⁴)2 or cyclopropyl; R² isfluorine; R³ is naphthalen-1-yl or naphthalen-2-yl; R⁴ is independentlyH, (C₁-C₃)-alkyl, (C₃-C₅)-cycloalkyl, or each R⁴ together with thenitrogen to which they are attached form

and n is 0 or 1.

In some embodiments of formula (I), R¹ is —N(R⁴)2 or cyclopropyl;

-   R² is independently halogen;-   R³ is naphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, or    isoquinolin-5-yl, wherein naphthalen-1-yl, naphthalen-2-yl,    quinolin-8-yl, or isoquinolin-5-yl is optionally independently    substituted with one or more fluorine atoms;-   R⁴ is independently H, (C₁-C₃)-alkyl, (C₃-C₅)-cycloalkyl, or each R⁴    together with the nitrogen to which they are attached form    NR⁶-piperazine, piperidine, pyrrolidine, azetidine, or morpholine;-   R⁶ is H, methyl or CD₃; and-   and n is an integer from 0-2.

In some embodiments of formula (I), R¹ is —N(CH₃)₂, cyclopropyl, or

-   R³ is naphthalen-1-yl or naphthalen-2-yl;-   R⁶ is H, methyl or CD₃; and-   n is0.

In some embodiments of formula (I), R¹ is

R³ is naphthalen-1-yl or naphthalen-2-yl; R⁶ is H, methyl or CD₃; and nis 0.

In some embodiments of formula (I), R¹ is

R³ is naphthalen-1-yl or naphthalen-2-yl; and n is 0.

In some embodiments of formula (I), the compound is

In some embodiments of formula (I), R¹ is —N(CH₃)₂ or cyclopropyl; R² isF; R³ is naphthalen-1-yl or naphthalen-2-yl; and n is 0 or 1.

In some embodiments of formula (I), R¹ is —N(CH₃)₂, cyclopropyl or

R² is halogen; R³ is naphthalen-1-yl or naphthalen-2-yl; R⁶ is H, methylor CD₃; and n is 1.

In some embodiments of formula (I), R¹ is —N(CH₃)₂, cyclopropyl or

R³ is naphthalen-1-yl or naphthalen-2-yl; R⁶ is H, methyl or CD₃; and nis 0.

In some embodiments of formula (I), R¹ is —N(CH₃)₂; R³ isnaphthalen-1-yl or naphthalen-2-yl; and n is 0.

In some embodiments of formula (I), R¹ is —N(CH₃)₂, cyclopropyl orN-methyl-piperazinyl; R² is fluoro; R³ is naphthalen-1-yl ornaphthalen-2-yl; and n is 1; or a pharmaceutically acceptable saltthereof.

In some embodiments of formula (I), R¹ is —N(CH₃)₂, cyclopropyl orN-methyl-piperazinyl; R³ is naphthalen-1-yl or naphthalen-2-yl; and n is0; or a pharmaceutically acceptable salt thereof.

In some embodiments of formula (I), the compound is

In some embodiments of formula (I), X₁ is N and X₂ is CH, or X₁ is CHand X₂ is N. In some embodiments of formula (I), X₁ is N and X₂ is CH.In some embodiments of formula (I), X₁ is CH and X₂ is N.

In some embodiments of formula (I), R¹ is —N(CH₃)₂, —NH(CH₃), orcyclopropyl; R² is independently halogen; R³ is naphthyl; and n is aninteger from 0-4; or a pharmaceutically acceptable salt thereof.

In some embodiments of formula (I), R¹ is —N(R⁴)₂, cyclopropyl, orR⁵-piperidin-4-yl. In some embodiments of formula (I), R¹ is —N(CH₃)₂,cyclopropyl, or

In some embodiments of formula (I), R¹ is

In some embodiments of formula (I), R¹ is

In some embodiments of formula (I), R¹ is —N(R⁴)₂, cyclopropyl, orN-methyl piperazinyl. In some embodiments of formula (I), R¹ is—N(CH₃)₂, cyclopropyl, or N-methyl piperazinyl. In some embodiments offormula (I), R¹ is —N(R⁴)₂ or cyclopropyl. In some embodiments offormula (I), R¹ is —N(CH₃)₂ or cyclopropyl. In some embodiments offormula (I), R¹ is —N(CH₃)₂ or —NH(CH₃). In some embodiments of formula(I), R¹ is cyclopropyl.

In some embodiments of formula (I), R² is independently D or halogen. Insome embodiments of formula (I), R² is halogen. In some embodiments offormula (I), R² is D. In some embodiments of formula (I), R² is chlorineor fluorine. In some embodiments of formula (I), R² is chlorine. In someembodiments of formula (I), R² is fluorine.

In some embodiments of formula (I), R³ is naphthyl, quinolinyl,isoquinolinyl, or indolyl, wherein said naphthyl, quinolinyl,isoquinolinyl or indolyl is optionally independently substituted with atleast one D, halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted withat least one D. In some embodiments of formula (I), R³ is naphthyl,quinolinyl, isoquinolinyl, or indolyl, wherein said naphthyl,quinolinyl, isoquinolinyl or indolyl is optionally independentlysubstituted with at least one D, halogen, (C₁-C₃)-alkoxy, or(C₁-C₃)-alkoxy substituted with at least one D; and wherein said indolylis not indol-4-yl. In some embodiments of formula (I), R³ is naphthyl,quinolinyl, isoquinolinyl, or indol-5-yl, wherein said naphthyl,quinolinyl, isoquinolinyl or indol-5-yl is optionally independentlysubstituted with at least one D, halogen, (C₁-C₃)-alkoxy, or(C₁-C₃)-alkoxy substituted with at least one D. In some embodiments offormula (I), R³ is naphthyl, quinolinyl, isoquinolinyl, or indolyl,wherein said naphthyl, quinolinyl, isoquinolinyl or indolyl isoptionally independently substituted with one or more halogen atoms or(C₁-C₃)-alkoxy. In some embodiments of formula (I), R³ is naphthyl,quinolinyl, isoquinolinyl, or indolyl, wherein said naphthyl,quinolinyl, isoquinolinyl or indolyl is optionally independentlysubstituted with one or more halogen atoms or (C₁-C₃)-alkoxy. In someembodiments of formula (I), R³ is naphthyl, quinolinyl, isoquinolinyl,or indol-5-yl, wherein said naphthyl, quinolinyl, isoquinolinyl orindol-5-yl is optionally independently substituted with one or morehalogen atoms or (C₁-C₃)-alkoxy. In some embodiments of formula (I), R³is naphthyl, quinolinyl, or isoquinolinyl, wherein said naphthyl,quinolinyl, or isoquinolinyl is optionally independently substitutedwith one or more halogen atoms or (C₁-C₃)-alkoxy. In some embodiments offormula (I), R³ is naphthyl, quinolinyl, or isoquinolinyl, wherein saidnaphthyl, quinolinyl, or isoquinolinyl is optionally independentlysubstituted with one or more halogen atoms. In some embodiments offormula (I), R³ is naphthyl, quinolinyl, or isoquinolinyl. In someembodiments of formula (I), R³ is naphthalen-1-yl, naphthalen-2-yl,quinolin-8-yl, isoquinolin-5-yl, or indol-5-yl, wherein saidnaphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, isoquinolin-5-yl, orindol-5-yl is optionally independently substituted with at least one D,halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least oneD. In some embodiments of formula (I), R³ is naphthalen-1-yl,naphthalen-2-yl, quinolin-8-yl, isoquinolin-5-yl, or indol-5-yl, whereinnaphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, isoquinolin-5-yl, orindol-5-yl is optionally independently substituted with one or morehalogen atoms or (C₁-C₃)-alkoxy. In some embodiments of formula (I), R³is naphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, isoquinolin-5-yl, orindol-5-yl. In some embodiments of formula (I), R³ is naphthalen-1-yl,naphthalen-2-yl, quinolin-8-yl, or isoquinolin-5-yl, whereinnaphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, or isoquinolin-5-yl isoptionally independently substituted with at least one halogen or(C₁-C₃)-alkoxy. In some embodiments of formula (I), R³ isnaphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, or isoquinolin-5-yl,wherein naphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, orisoquinolin-5-yl, is optionally independently substituted with one ormore halogen atoms. In some embodiments of formula (I), R³ isnaphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, or isoquinolin-5-yl,wherein naphthalen-1-yl, naphthalen-2-yl, quinolin-8-yl, orisoquinolin-5-yl is optionally independently substituted with onefluorine atom. In some embodiments of formula (I), R³ is naphthalen-1-ylor naphthalen-2-yl, optionally substituted with one or more halogenatoms. In some embodiments of formula (I), R³ is naphthalen-1-yl ornaphthalen-2-yl.

In some embodiments of formula (I), R³ is

In some embodiments of formula (I), R³ is

In some embodiments of formula (I), R³ is

In some embodiments of formula (I), R³ is

In some embodiments of formula (I), R³ is

In some embodiments of formula (I), R³ is naphthyl, quinolinyl orisoquinolinyl.

In some embodiments of formula (I), R³ is naphthyl, independentlysubstituted with one or more halogen atoms. In some embodiments offormula (I), R³ is naphthalen-1-yl or naphthalen-2-yl, independentlysubstituted with one or more halogen atoms. In some embodiments offormula (I), R³ is quinolinyl, independently substituted with one ormore halogen atoms. In some embodiments of formula (I), R³ isquinolinyl. In some embodiments of formula (I), R³ is quinolin-8-yl,independently substituted with one or more halogen atoms. In someembodiments of formula (I), R³ is quinolin-8-yl. In some embodiments offormula (I), R³ is isoquinolinyl, independently substituted with one ormore halogen atoms. In some embodiments of formula (I), R³ isisoquinolinyl. In some embodiments of formula (I), R³ isisoquinolin-5-yl, independently substituted with one or more halogenatoms. In some embodiments of formula (I), R³ is isoquinolin-5-yl. Insome embodiments of formula (I), R³ is indolyl. In some embodiments offormula (I), R³ is indolyl, independently substituted with one or morehalogen atoms. In some embodiments of formula (I), R³ is indol-5-yl,independently substituted with one or more halogen atoms. In someembodiments of formula (I), R³ is indol-5-yl.

In some embodiments of formula (I), R³ is independently substituted withone or more halogen atoms. In some embodiments of formula (I), R³ isindependently substituted with one or more D. In some embodiments offormula (I), R³ is independently substituted with one D.

In some embodiments of formula (I), the halogen atom is chlorine orfluorine. In some embodiments of formula (I), the halogen atom ischlorine. In some embodiments of formula (I), halogen atom is fluorine.

In some embodiments of formula (I), R⁴ is independently H,(C₁-C₄)-alkyl, (C₁-C₄)-alkyl substituted with at least one D,(C₃-C₅)-cycloalkyl, or each R⁴ together with the nitrogen to which theyare attached form a 3-7 membered heterocyclic ring, wherein one of thecarbon atoms is optionally replaced with NR⁶, O or S. In someembodiments of formula (I), R⁴ is independently H, (C₁-C₃)-alkyl,(C₁-C₃)-alkyl substituted with at least one D, (C₃-C₅)-cycloalkyl, oreach R⁴ together with the nitrogen to which they are attached form a 3-7membered heterocyclic ring, wherein one of the carbon atoms isoptionally replaced with NR⁶, O or S. In some embodiments of formula(I), R⁴ is independently H, (C₁-C₃)-alkyl, (C₃-C₅)-cycloalkyl, or eachR⁴ together with the nitrogen to which they are attached form a 3-7membered heterocyclic ring, wherein one of the carbon atoms isoptionally replaced with NR⁶, O or S. In some embodiments of formula(I), R⁴ is independently H, (C₁-C₃)-alkyl, (C₃-C₅)-cycloalkyl, or eachR⁴ together with the nitrogen to which they are attached form N-methylpiperazine, piperazine, piperidine, pyrrolidine, azetidine, ormorpholine. In some embodiments of formula (I), R⁴ is independently H,(C₁-C₃)-alkyl, or each R⁴ together with the nitrogen to which they areattached form

In some embodiments of formula (I), each R⁴ together with the nitrogento which they are attached form

In some embodiments of formula (I), R⁴ is independently H or methyl. Insome embodiments of formula (I), R⁴ is independently H, methyl or ethyl.In some embodiments of formula (I), R⁴ is independently propyl or butyl.In some embodiments of formula (I), R⁴ is independently methyl or ethyl.In some embodiments of formula (I), R⁴ is independently methyl. In someembodiments of formula (I), R⁴ is independently ethyl.

In some embodiments of formula (I), R⁴ is independently H or CD₃. Insome embodiments of formula (I), R⁴ is independently H, CD₃ or CH₂CD₃.In some embodiments of formula (I), R⁴ is independently CD₃ or CH₂CD₃.In some embodiments of formula (I), R⁴ is independently CD₃. In someembodiments of formula (I), R⁴ is independently CH₂CD₃.

In some embodiments of formula (I), R⁵ is H or (C₁-C₃)-alkyl, or(C₁-C₃)-alkyl substituted with at least one D. In some embodiments offormula (I), R⁵ is H or (C₁-C₃)-alkyl. In some embodiments of formula(I), R⁵ is H or methyl. In some embodiments of formula (I), R⁵ is H. Insome embodiments of formula (I), R⁵ is H, methyl or ethyl. In someembodiments of formula (I), R⁵ is methyl or ethyl. In some embodimentsof formula (I), R⁵ is methyl. In some embodiments of formula (I), R⁵ isethyl.

In some embodiments of formula (I), R⁵ is H or CD₃. In some embodimentsof formula (I), R⁵ is H, CD₃ or CH₂CD₃. In some embodiments of formula(I), R⁵ is CD₃ or CH₂CD₃. In some embodiments of formula (I), R⁵ is CD₃.In some embodiments of formula (I), R⁵ is CH₂CD₃.

In some embodiments of formula (I), R⁶ is H or (C₁-C₃)-alkyl, or(C₁-C₃)-alkyl substituted with at least one D. In some embodiments offormula (I), R⁶ is H or (C₁-C₃)-alkyl. In some embodiments of formula(I), R⁶ is H or methyl. In some embodiments of formula (I), R⁶ is H. Insome embodiments of formula (I), R⁶ is H, methyl or ethyl. In someembodiments of formula (I), R⁶ is methyl or ethyl. In some embodimentsof formula (I), R⁶ is methyl. In some embodiments of formula (I), R⁶ isethyl.

In some embodiments of formula (I), R⁶ is H, methyl or CD₃. In someembodiments of formula (I), R⁶ is H or CD₃. In some embodiments offormula (I), R⁶ is H, CD₃ or CH₂CD₃. In some embodiments of formula (I),R⁶ is CD₃ or CH₂CD₃. In some embodiments of formula (I), R⁶ is CD₃. Insome embodiments of formula (I), R⁶ is CH₂CD₃.

In some embodiments of formula (I), n is an integer from 0-4. In someembodiments of formula (I), n is an integer from 0-3. In someembodiments of formula (I), n is an integer from 0-2. In someembodiments of formula (I), n is an integer from 0-1. In someembodiments of formula (I), n is an integer from 1-2. In someembodiments of formula (I), n is 0. In some embodiments of formula (I),n is 1. In some embodiments of formula (I), n is 2.

In some embodiments, R³ is not indolyl. In some embodiments, R³ is notindolyl when n is 0. In some embodiments, R³ is not indol-4-yl. In someembodiments, R³ is not indol-4-yl when n is 0. In some embodiments, R⁴is not H or (C₁-C₃)-alkyl when R³ is indolyl and n is 0. In someembodiments, when R³ is indol-4-yl and n is 0, R¹ is notN-methyl-piperazinyl.

Abbreviations and Definitions

The term “compound of the invention” as used herein means a compound offormula (I) or any subgenus or species thereof. The term is alsointended to encompass salts, hydrates, and solvates thereof.

The term “composition(s) of the invention” as used herein meanscompositions comprising a compound of the invention. The compositions ofthe invention may further comprise other agents such as, for example,carriers, excipients, stabilants, lubricants, solvents, and the like.

The term “D” refers to a deuterium atom, and is known in the art torefer to a deuterium enriched species, that is, where D is present aboveits natural isotopic abundance.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds of the invention with other chemical components, such asphysiologically acceptable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of a compoundto an organism or subject.

The term “pharmaceutically acceptable salt” is intended to include saltsderived from inorganic or organic acids including, for examplehydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric,formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic,salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic,trifluroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids;and salts derived from inorganic or organic bases including, for examplesodium, potassium, calcium, ammonium or tetrafluoroborate. Exemplarypharmaceutically acceptable salts are found, for example, in Berge, etal. (J. Pharm. Sci. 1977, 66(1), 1; hereby incorporated by reference inits entirety).

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Non-limiting examples of suchpharmaceutical carriers include liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical carriers may also be saline, gum acacia, gelatin,starch paste, talc, keratin, colloidal silica, urea, and the like. Inaddition, auxiliary, stabilizing, thickening, lubricating and coloringagents may be used. Other examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences (Alfonso Gennaroed., Krieger Publishing Company (1997); Remington's: The Science andPractice of Pharmacy, 21^(st) Ed. (Lippincot, Williams & Wilkins (2005);Modern Pharmaceutics, vol. 121 (Gilbert Banker and Christopher Rhodes,CRC Press (2002); each of which hereby incorporated by reference in itsentirety).

In some embodiments of the compound of formula (I), R¹ is —N(CH₃)₂ or—NH(CH3); R³ is

and n is 0.

In some embodiments of the compound of formula (I), R¹ is —N(CH₃)₂; R³is

and n is 0.

In some embodiments of the compound of formula (I), R¹ is —N(CH₃)₂; R³is

and n is 0.

In some embodiments of the compound of formula (I), R¹ is —N(CH₃)₂; R³is

and n is 0.

In some embodiments of formula (I), R¹ is cyclopropyl; R² is halogen;and R³ is

In some embodiments of formula (I), R¹ is cyclopropyl; R² is halogen;and R³ is

In some embodiments of formula (I), R¹ is cyclopropyl; R² is halogen;and R³ is

In some embodiments of formula (I), R¹ is cyclopropyl; R² is fluorine;R³ is

and n is 1.

In some embodiments of formula (I), R¹ is cyclopropyl; R² is fluorine;R³ is

and n is 1.

In some embodiments of formula (I), the compound is selected from

In some embodiments of formula (I), the compound is selected from

In some embodiments of formula (I), the compound is

In some embodiments of formula (I), the compound is selected from

In some embodiments of formula (I), the compound is selected from

In some embodiments of formula (I), the compound is

also referred to herein is (1) or MW-181).

In some embodiments of formula (I), the compound is

also referred to herein is (2) or MW-108).

In some embodiments of formula (I), the compound is

also referred to herein is (7) or MW-077).

In some embodiments of formula (I), the compound is

also referred to herein is (9) or MW-125).

In some embodiments of formula (I), the compound is

also referred to herein is (10) or MW-167).

In some embodiments of formula (I), the compound is

also referred to herein is (27) or MW-150).

In some embodiments of formula (I), the compound is

also referred to herein is (17) or MW-078).

In some embodiments of formula (I), the compound is

also referred to herein is (18) or MW-085).

In some embodiments of formula (I), the compound is

also referred to herein is (20) or MW-082).

In some embodiments of formula (I), the compound is

also referred to herein is (21) or MW-165).

In some embodiments of formula (I), the compound is

also referred to herein is (27) or MW-150).

In some embodiments, the compound is selected from the compounds listedin Table 1. In some embodiments, the compound is a compound of Table 1.

TABLE 1 Representative MAPK Modulators.           Compound

          R³           R¹  1 (MW-181)

—N(CH₃)₂  2 (MW-108)

—N(CH₃)₂  7 (MW-077)

 9 (MW-125)

10 (MW-167)

11 (MW-122)

12 (MW-124)

13 (MW-107)

14 (MW-109)

15 (MW-156)

—NHCH₃ 16 (MW-200)

—N(H)CH₃ 17 (MW-078)

—N(CH₃)₂ 18 (MW-085)

—N(CH₃)₂ 19 (MW-155)

—N(CH₃)₂ 20 (MW-082)

—N(CH₃)₂ 21 (MW-165)

—N(CH₃)₂ 22 (MW-066)

—N(CH₃)₂ 23 (MW-033)

—N(CH₂CH₃)₂ 24 (MW-010)

25 (MW-031)

—N(H)CH(CH₃)₂ 26 (MW-025)

—N(H)CH(CH₃)₂ 27 (MW-150)

28 (MW-118)

30 (MW-126)

31 (MW-146)

32 (MW-148)

33 (MW-152)

34 (MW-154)

35 (MW-153)

36 (MW-164)

37 (MW-149)

45

46 (SRM-137C)

47

48

49 (MW-203)

50 (MW-017)

51 (MW-044)

52 (MW-032)

53 (MW-059)

54 (MW-197)

55 (MW-063)

56 (SRM-137A)

57 (SRM-137B)

59 (SRM-203A)

60 (SRM-203B)

61 (MW-086)

62 (MW-026)

64

65

66

—N(CH₃)₂ 67

68

69

70

71

72

73

—N(CH₃)₂ 74

75

76

77

—N(CH₃)₂ 78

79

80

44 (MW-064)

—N(CH₃)₂ 63 (SRM-138B)

In another aspect, the invention is directed to compositions comprisinga compound of formula (I) and a pharmaceutically acceptable carrier.

In another aspect, the invention is directed to a method of inhibitingp38α MAPK comprising contacting MAPK with a compound of formula (I). Insome embodiments, the compound of formula (I) exhibits a Ki for p38αMAPK of less than about one micromolar. In some embodiments, thecompound of formula (I) exhibits a Ki for p38α MAPK of less than about500 nM. In some embodiments, the compound of formula (I) exhibits a Kifor p38α MAPK of less than about 200 nM. In some embodiments, thecompound of formula (I) exhibits a Ki for p38α MAPK of less than about100 nM. In some embodiments, the compound of formula (I) exhibits a Kifor p38α MAPK of less than about one micromolar.

In another aspect, the invention is directed to a method of treatingdiseases or disorders where MAPK is over-expressed or over-activated. Insome embodiments, such diseases include, for example, inflammatorydiseases, neurodegenerative diseases, skin disorders, etc. Otherdisorders related to MAPK are described, for example, in U.S. Pat. No.7,919,485; EP Patent Nos. 1196167, 1606283, and 2426134; U.S. PatentPublication Nos. 2010/0104536 and 20120289511; Nat. Rev. Drug Disc.2003, 2, 717; Curr. Opin, Drug Disc. Dev. 2005, 8, 421; Exp. Opin. Ther.Patents 2011, 21, 1843; J. Neuroinflammation 2007, 4, 21; andNeuropharmacology 2010, 58, 561; each herein incorporated by referencein its entirety.

In another aspect, the invention is directed to a method of treatingdisease in a subject comprising administration of a therapeuticallyeffective amount of a compound of formula (I). In some embodiments, thedisease is characterized by inflammation. In some embodiments, thedisease comprises ischemia. In some embodiments, the disease is aneurological disorder. In some embodiments, the disease is aneurodegenerative disease. In some embodiments, the neurodegenerativedisease is Huntington's Disease, Parkinson's Disease, multiplesclerosis, ALS or Alzheimer's Disease. In some embodiments, theneurodegenerative disease is Huntington's Disease, Parkinson's Disease,ALS or Alzheimer's Disease. In some embodiments, the disease isAlzheimer's Disease.

In another aspect, the invention is directed to a method of treatingneurodegenerative disease in a subject comprising administration of atherapeutically effective amount of a composition comprising a compoundof formula (I). In some embodiments, the disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of increasinglong-term potentiation in a subject having a neurodegenerative diseasecomprising administration of a therapeutically effective amount of acompound of formula (I). In some embodiments, the neurodegenerativedisease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of increasinglong-term potentiation in a subject having a neurodegenerative diseasecomprising administration of a therapeutically effective amount of acomposition comprising a compound of formula (I). In some embodiments,the neurodegenerative disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingmemory in a subject having a neurodegenerative disease comprisingadministration of a therapeutically effective amount of a compound offormula (I). In some embodiments, the neurodegenerative disease isAlzheimer's Disease.

In another aspect, the invention is directed to a method of improvingmemory in a subject having a neurodegenerative disease comprisingadministration of a therapeutically effective amount of a compositioncomprising a compound of formula (I). In some embodiments, theneurodegenerative disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingsynaptic function in a subject having a neurodegenerative diseasecomprising administration of a therapeutically effective amount of acompound of formula (I). In some embodiments, synaptic functioncomprises synaptic plasticity. In some embodiments, synaptic plasticitycomprises learning, memory, or a combination thereof. In someembodiments, synaptic plasticity comprises long term potentiation (LTP).In some embodiments, the neurodegenerative disease is Alzheimer'sDisease.

In another aspect, the invention is directed to a method of improvingsynaptic function in a subject having a neurodegenerative diseasecomprising administration of a therapeutically effective amount of acomposition comprising a compound of formula (I). In some embodiments,synaptic function comprises synaptic plasticity. In some embodiments,synaptic plasticity comprises learning, memory, or a combinationthereof. In some embodiments, synaptic plasticity comprises long termpotentiation (LTP). In some embodiments, the neurodegenerative diseaseis Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingsynaptic function in a subject having ischemia comprising administrationof a therapeutically effective amount of a composition comprising acompound of formula (I). In some embodiments, the subject has aneurodegenerative disease. In some embodiments, the neurodegenerativedisease ALS, Huntington's Disease, Parkinson's Disease or Alzheimer'sDisease. In some embodiments, the ischemia is brain ischemia.

Another aspect of the invention provides a method for increasing memoryretention in a subject afflicted with a neurodegenerative disease, themethod comprising administering to a subject a therapeutic amount of acompound of formula (I) or a composition comprising a compound offormula (I).

In some embodiments, a compound of formula (I) is administered. In someembodiments, a composition comprising a compound of formula (I) isadministered.

Exemplary neurodegenerative diseases comprise Adrenoleukodystrophy(ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer'sdisease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxiatelangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases ProgressiveSupranuclear Palsy, Rett's syndrome, Tau-positive FrontoTemporaldementia, Tau-negative FrontoTemporal dementia, Refsum's disease,Sandhoff disease, Schilder's disease, Subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),Spinocerebellar ataxia (multiple types with varying characteristics),Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabesdorsalis, or Toxic encephalopathy. Exemplary neurodegenerative diseasesand methods of treatment therefor are also described in WO 2010/074783,WO 2011/072243, and WO 2012/088420; each herein incorporated byreference in its entirety.

In some embodiments, the disease is characterized by altered cellularfunction, synaptic dysfunction, fast axonal transport, neuraldegeneration, or neuroinflammation.

In some embodiments, the disorder or disease is Alzheimer's disease,Parkinson's disease, Huntington disease, Down syndrome, head trauma,traumatic brain injury (TBI), brain injury due to cerebral focalischemia, attention deficit disorder, neuronal degeneration with brainiron accumulation type I (Hallervorden-Spatz disease), Lytico-Bodigdisease (Parkinson-dementia complex of Guam), pure autonomic failure,REM sleep behavior disorder, mild cognitive impairment (MCI), cerebralamyloid angiopathy (CAA), mild cognitive deficits, aging, vasculardementias mixed with Alzheimer's disease, any neurodegenerative diseasecharacterized by abnormal amyloid deposition, or any combinationthereof, Adrenoleukodystrophy (ALD), Alcoholism, Alexander's disease,Alper's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease),Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Rett's syndrome, Tau-positive FrontoTemporaldementia, Tau-negative FrontoTemporal dementia, Dementia pugilistica(chronic traumatic encephalopathy), Refsum's disease, Sandhoff disease,Schilder's disease, Subacute combined degeneration of spinal cordsecondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease(also known as Batten disease), Spinocerebellar ataxia (multiple typeswith varying characteristics), Spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, or Toxicencephalopathy, Inclusion body myositis, or neuroinflammation.Neuroinflammation has been also found to play a role in psychiatricillnesses. Indeed, a link between psychiatric symptoms and autoimmunityin autoimmune diseases, neuroimmunological abnormalities occur inclassical psychiatric disorders (for example, major depressive, bipolar,schizophrenia, and obsessive compulsive disorders).

In some embodiments, the neurodegenerative disease comprisesAdrenoleukodystrophy (ALD), Alcoholism, Alexander's disease, Alper'sdisease, Alzheimer's disease, Amyotrophic lateral sclerosis (LouGehrig's Disease), Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases ProgressiveSupranuclear Palsy, Rett's syndrome, Tau-positive FrontoTemporaldementia, Tau-negative FrontoTemporal dementia, Refsum's disease,Sandhoff disease, Schilder's disease, Subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),Spinocerebellar ataxia (multiple types with varying characteristics),Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabesdorsalis, or Toxic encephalopathy.

In some embodiments, the neurodegenerative disease comprises Alzheimer'sdisease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease),Huntington's disease, or Parkinson's disease.

In some embodiments, the neurodegenerative disease is Alzheimer'sdisease. In some embodiments, the neurodegenerative disease isAmyotrophic lateral sclerosis. In some embodiments, theneurodegenerative disease is Huntington's disease. In some embodiments,the neurodegenerative disease is Parkinson's disease.

Compounds of formula (I) can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions can comprisea compound of formula (I) and a pharmaceutically acceptable carrier.Thus, in some embodiments, the compounds of the invention are present ina pharmaceutical composition.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

Any of the therapeutic applications described herein can be applied toany subject in need of such therapy, including, for example, a mammalsuch as a mouse, a rat, a dog, a cat, a cow, a horse, a rabbit, amonkey, a pig, a sheep, a goat, or a human. In some embodiments, thesubject is a mouse, rat or human. In some embodiments, the subject is amouse. In some embodiments, the subject is a rat. In some embodiments,the subject is a human.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyethylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, itcan be useful to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of useful preparation methods arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

Pyridazine and pyrazine based compounds are synthesized by methodswithin the purview of the ordinarily skilled artisan. Exemplary methodsby which such derivatives can be synthesized are as follows, in additionto those described in, for example, U.S. Pat. Nos. 8,063,047; 8,158,627;8,188,096; and 8,367,672; and Bioorg. Med. Chem. Lett. 2007, 17, 414;each herein incorporated by reference in its entirety. Additionalexemplary methods of preparation for pyridazines and pyrazines are shownin Scheme A and the Examples.

Initially, the starting anhydride A can be treated with hydrazine suchas hydrazine sulfate in water and heated to about 100° C. for about 18hours to provide B. Compound B can then be cross-coupled with a pyridylderivative such as a pyridyl boronic acid using exemplary reagentsPd(PPh₃)₄, a carbonate base such as sodium carbonate in a solvent systemsuch as DME/water following typical cross-coupling procedures to provideC. Compound C can then be treated with a chlorine source such as POCl₃in a solvent such as acetonitrile at elevated temperature such as about90° C. for about 18 hours to provide D. Compound D can then be treatedwith an amine (HNRR′) at elevated temperature, such as about 120° C. forabout 12-18 hours to provide E. Compound E can be treated with aarylboronic acid or heteroaryl boronic acid under standardcross-coupling conditions using exemplary reagents such as reagentsPd(PPh₃)₄, a carbonate base such as sodium carbonate in a solvent systemsuch as DME/water to provide compounds F.

Deuterium enriched compounds of formula (I) can also be synthesizedusing known intermediates and/or starting materials. For example,compounds containing a CD₃ can be made by incorporating building blockssuch as CD₃I into the exemplary syntheses herein. Deuterium enrichedaryl intermediates can also be substituted for non-deuterium enrichedaryl intermediates in the methods described herein to obtain compoundsof formula (I) comprising deuterium enriched aryl groups. For example,deuterated naphthyl boronic acid or deuterated naphthyl carboxylic acidscan be used to generate compounds of formula (I) wherein R³ comprises adeuterated naphthyl group.

It will recognized that one or more features of any embodimentsdisclosed herein may be combined and/or rearranged within the scope ofthe invention to produce further embodiments that are also within thescope of the invention. Those skilled in the art will recognize, or beable to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be within the scope of thepresent invention.

The invention is further described by the following non-limitingExamples.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are illustrative only, since alternative methods can beutilized to obtain similar results.

Chemicals were purchased from Aldrich (Milwaukee, Wis.) or VWRInternational (Batavia, Ill.). Solvents were used as received unlessstated otherwise. Water was obtained using a Milli-Q Biocel A10purification system from Millipore Corporation (Bedford, Mass.). Highpressure glassware was from Chemglass, (Vineland, N.J.). All syntheseswere monitored by HPLC (Dionex System, Sunnyvale, Calif., with UVD170Uultraviolet detector and P680 pump) on a Phenomenex (Torrance, Calif.)Luna C18 column (250×2.0 mm; 5 μm; equipped with guard column) at a flowrate of 0.2 mL/min and using a mobile phase composed of 0.1% (v/v)formic acid (Fluka) in water as reagent A and 80% acetonitrile, 0.08%formic acid/water as reagent B. Synthetic intermediates were screened byelectrospray mass spectroscopy (ESI) HPLC (Micromass Quattro II TripleQuadrupole HPLC/MS/MS mass spectrometer). Final compounds were analyzedby HPLC, high-resolution mass spectra (HRMS; VG70-250SE massspectrometer) and 1H NMR (Bruker Avance-III 500 MHz spectrometer;ambient temperature). Melting point data for compounds were acquired ona Büchi Melting Point B-540 (Flawil, Switzerland). Hydrochloridehydrates (salts) of final compounds were characterized by elementalanalysis (Intertek QTI; Whitehouse, N.J.).

Abbreviations used are: MeOH=methanol, CDCl₃=deuterated chloroform,CD₃OD=deuterated methanol, THF=tetrahydrofuran, DME=1,2-dimethoxyethane,POBr₃=phosphorus oxybromide, POCl3=phosphorus oxychloride, Na₂CO₃=sodiumcarbonate, EtOAc=ethyl acetate, LDA=lithium diisopropylamide,DIEA=diisopropylethylamine, TEBAC=tetraethylbutyl ammonium chloride,DMSO=dimethylsulphoxide.

HPLC Method A: Gradient—0% to 100% B, 15 min; isocratic 22 min. Forpurity analysis by HPLC, compounds were analyzed at ˜0.50 μg level andpeak quantification was performed based upon Relative area % absorptionat 260 nm

LC/MS method A: LC/MS data were determined with a Waters Alliance 2695HPLC/MS (Waters Symmetry C18, 4.6×75 mm, 3.5 lm) with a 2996 diode arraydetector from 210-400 nm; the solvent system is 5-95% MeCN in water(with 0.1% TFA) over nine mins using a linear gradient and retentiontimes are in minutes. Mass spectrometry was performed on a Waters ZQusing electrospray in positive mode.

HPLC method B: Preparative reversed phase chromatography was performedon a Waters Sunfire column (19×50 mm, C18, 5 micron) with a mobile phaseof 10% acetonitrile/water to 90% acetonitrile/water with 0.1% TFA asbuffer using 214 nm and 254 nm as detection wavelengths. Injection andfraction collection were performed with a Gilson 215 liquid handlingapparatus using Trilution LC software.

Animals: All experiments were conducted in accordance with theprinciples of animal care and experimentation in the Guide For the Careand Use of Laboratory Animals. The Institutional Animal Care and UseCommittees of the three universities approved the use of animals in thisstudy. For efficacy experiments, 3- to 4-month-old C57BL/6J male micefrom a colony bred in the Columbia University animal facility were used.At the University of Kentucky, the in vivo LPS experiment was done with2-month-old, female, C57Bl/6J mice from Jackson Laboratory. The p38αMAPK drug-resistant knock-in (p38α^(T106M)) mice were generated byreplacement of Thr106 in p38α with Met as described in J. Biol. Chem.2007, 282, 34663 (herein incorporated by reference in its entirety).Remaining rodent studies were done at Northwestern University aspreviously described (J. Neuroinflamm. 2007, 4, 21; Bioorg. Med. Chem.Lett. 2007, 17, 414; each herein incorporated by reference in itsentirety).

Example 1: Synthesis of MW-181 (1) and MW-108 (2)

4-bromo-1,2-dihydropyridazine-3,6-dione (1-1): Hydrazine sulfate (2.25g, 17.2 mmol) was dissolved in boiling water (20 mL) with stirring. Tothis solution, bromomaleic anhydride (2.6 mL, 28.2 mmol) was addeddropwise via addition funnel, the mixture heated (100° C.) under refluxfor 19 h, then cooled to ambient temperature. The resulting whiteprecipitate was filtered on a medium frit sintered glass funnel, washedwith acetone (3×5 ml), and air dried in vacuo to give the desiredproduct 4-bromo-1,2-dihydropyridazine-3,6-dione (2.85 g) as a whitepowder in 87% yield (gravimetric) with a melting point of 262° C.

4-(pyridin-4-yl)-1,2-dihydropyridazine-3,6-dione (1-2): Essentially aspreviously described (J. Neuroinflamm. 2007, 4, 21; herein incorporatedby reference in its entirety), compound 1-1 (2 g, 10.4 mmol, 1 eq) andpyridin-4-yl boronic acid (14.3 mmol, 1.76 g, 1.37 eq) were suspended indimethoxyethane and water (10:1 v/v) in a heavy wall pressure vessel andpurged with argon for 15 min. Tetrakis(triphenylphosphine)palladium (0.1eq) and sodium carbonate (3 eq) were added, the vessel immediatelycapped, the reaction mixture heated (110° C.) for 18 h, then cooled toambient temperature and subjected to filtration on a medium fritsintered glass funnel containing Celite® 545. The filtrate wasconcentrated in vacuo and the concentrate triturated with hexane. Theyellow product 1-2 (2.2 g) exhibited a mass (ESI) of m/z (MeOH)=190.06(MH⁺), and was taken to the next step without further purification.

3,6-dichloro-4-(pyridin-4-yl)pyridazine (1-3): Essentially as described(Bioorg. Med. Chem. Lett. 2007, 17, 414; herein incorporated byreference in its entirety), compound 1-2 (2.2 g, 11.6 mmol) wassuspended in 6.25 mL phosphorus oxychloride in a condenser-fitted roundbottom flask, heated (90° C.) for 24 h, cooled to ambient temperatureand volatiles removed in vacuo. The dark residue was poured onto crushedice, stirred (2 h), and the mixture neutralized with saturated sodiumcarbonate solution. The fine precipitate was subjected to replicateextraction with dichloromethane in a separatory funnel, the combinedorganic phases subjected to drying over anhydrous sodium sulfate,concentrated in vacuo, and subjected to column chromatography on silicagel (200-400 mesh) using a ethyl acetate:hexane (3:2 v/v) eluent. Thedesired product 1-3 exhibited 97% purity by HPLC and a mass (ESI) of m/z(MeOH)=225.99 (MH⁺). The overall yield (gravimetric) from product 1-1 to1-3 was approximately 36%.

6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (1-4): Followingpublished protocol (Bioorg. Med. Chem. Lett. 2007, 17, 414; hereinincorporated by reference in its entirety), compound 1-3 (11.06 mmol, 1eq) in ethanol (60 mL) was placed in a heavy wall pressure vessel and40% dimethylamine (6 eq) added, the vessel capped and heated (110° C.)for 16 h, the mixture allowed to cool to ambient temperature thentransferred to a round bottom flask for concentration in vacuo. Theresidue was treated with 10 mL of water, the aqueous phase subjected toreplicate extraction with dichloromethane in a separatory funnel, andthe combined organic layers dried (anhydrous sodium sulfate) andevaporated under reduced pressure to yield a yellow adherent solid. Thereaction mixture was purified by silica gel (200-400 mesh) columnchromatography and the desired product eluted with ethyl acetate:hexane(1:1 v/v). Product 1-4 was obtained as a yellow solid in 89%(gravimetric) yield, with an apparent HPLC purity of 98% and a mass(ESI) m/z (MeOH)=235.10 (MH⁺).

N,N-dimethyl-6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-amine(1=MW-181): Compound 1-4 (1.95 g, 8.5 mmol, 1 eq) and 1-naphthylboronicacid (1.96 g, 11.3 mmol, 1.37 eq) were suspended in dimethoxyethane(DME) and water (10:1, v/v) in a heavy wall pressure vessel and purgedwith argon for 15 min. Tetrakis(triphenylphosphine)palladium (0.860 g, 9mol %) and Na₂CO₃ (2.7 g, 25.7 mmol, 3.1 eq) were added, the vesselpurged with argon and immediately capped with a Teflon bushing, heated(110° C.) for 17 hr, and cooled to ambient temperature. The reactionmixture was subjected to filtration on a medium frit sintered glassfunnel containing Celite® 545. The resultant filtrate was concentratedin vacuo, the residue dissolved in CH₂Cl₂, and subjected to waterextraction (3×30 mL) in a separatory funnel. The organic layer was driedover anhydrous sodium sulfate and concentrated by rotary evaporationunder reduced pressure. The crude mixture was subjected to silica gel(200-400 mesh) column chromatography with product elution using ethylacetate: hexane (2:3 v/v). The product was crystallized from hexane andethyl acetate. Product 1 (MW-181) was obtained as light yellow crystalsin 56% yield (gravimetric). HPLC purity, 98%; ESI m/z (MeOH), 327.10(MH⁺); MP=189.5-190° C. (uncorrected); HRMS, 326.1526 (calculated forC₂₁H₁₈N₄=326.1531). ¹H-NMR (500 MHz, CDCl₃): δ 8.34 (d, J=5.8 Hz, 2H);7.83 (d, J=8.2 Hz, 2H); 7.68 (d, J=8.4 Hz, 1H); 7.42-7.26 (m, 4H); 6.99(dd, J=1.5, 4.6, 2H); 6.84 (s, 1H); 3.33 (s, 6H).

N, N-dimethyl-6-(naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3-amine(2=MW-108): Product 2 was produced using the protocol as above for 1 butusing 2-naphthylboronic acid (595 mg, 2.9 mmol, 1.37 eq) suspended inDME:water mixture (10:1, v/v). The final product, 2 (MW-108) wasobtained as a whitish powder in 52% yield (gravimetric). HPLC purity,96%; ESI m/z (MeOH), 327.10 (MW); MP=181.5-182° C. (uncorrected); HRMS,326.1538 (calculated for C₂₁H₁₈N₄=326.1531). ¹H-NMR (500 MHz, CDCl₃): δ8.55 (d, J=5.65 Hz, 2H); 7.93 (s, 1H); 7.80-7.68 (m, 3H); 7.49-7.34 (m,3H); 7.19 (d, J=5.95 Hz, 2H); 6.76 (s, 1H); 3.30 (s, 6H).

Hydrochloride hydrates of products 1 and 2: This was done as described(Bioorg. Med. Chem. Lett. 2007, 17, 414; herein incorporated byreference in its entirety). Approximately 1 mmol of the respectivecompound was suspended in 8.2 mL of anhydrous isopropanol (99.5%,Aldrich), heated to 85° C. with stirring until dissolved, then 3.1 equiv(234 μL) of concentrated ultrapure HCl (12N, JT Baker Ultrex® II,Product 6900-05) was added to the clear solution, resulting in immediategeneration of a suspension of solids. The suspension was stirred for 10min (80° C.), allowed to cool to ambient temperature, then the vesselplaced on ice for ˜2.5 h, then stored (˜16 hr) at 4° C. The resultingyellow precipitate was filtered on a medium frit sintered glass funnelunder vacuum, washed (3×) with cold anhydrous isopropanol followed bythree washes with cold anhydrous ether, and dried under vacuum. Theprecipitate was stored in vacuo in a glass desiccator containing silicagel until a constant weight was attained. The final products wereobtained in approximately 82% yield (gravimetric) compared to thestarting material. Hydrochloride hydrate formation was confirmed byelemental analysis. Elemental analysis indicated a ratio of ˜2 HCl:compound. EA calculated for C₂₁H₂₄Cl₂N₄O₂ is: C, 57.94; H, 5.56; N,12.87; Cl, 16.29; O, 7.35. Experimentally found for MW-181: C, 58.16; H,5.60; N, 12.51; Cl, 16.52; O, 7.37. Experimentally found for MW-108: C,56.09; H, 5.65; N, 12.38; Cl, 15.91; O, 10.16.

Example 1-1

Compound 57(6-(4-methylpiperidin-1-yl)-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine)(also described herein as SRM-137B) can be prepared in a similar manneras described in Example 1 by using 4-methylpiperidine as the amine andby using 1-naphthylboronic acid instead of 2-naphthylboronic acid.

Example 1-2

Compound 59(3-(naphthalen-1-yl)-4-(pyridin-4-yl)-6-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine)(also described herein as SRM-203A) can be prepared in a similar manneras described in Example 1 by using 2-(piperazin-1-yl)pyrimidine as theamine and by using 1-naphthylboronic acid instead of 2-naphthylboronicacid.

Example 1-3

Compound 56(6-(4-methylpiperidin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine)(also described herein as SRM-137A) can be prepared in a similar manneras described in Example 1 by using 4-methylpiperidine as the amine andby using 2-naphthylboronic instead of 1-naphthylboronic acid.

Example 1-4

Compound 60(3-(naphthalen-2-yl)-4-(pyridin-4-yl)-6-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine)(also described herein as SRM-203B) can be prepared in a similar manneras described in Example 1 by using 2-(piperazin-1-yl)pyrimidine as theamine and by using 2-naphthylboronic instead of 1-naphthylboronic acid.

Example 2: Synthesis of Amino-Pyridazine Analogues

Amino-pyridazine analogues described herein are made via approachesdescribed in Bioorg. Med. Chem. Lett. 2007, 17, 414; herein incorporatedby reference in its entirety.

1-Phenyl-2-(pyridin-4-yl)ethanone (2-1): THF in a round bottom flask wascooled (−78° C.), lithium diisopropylamide added to the flask withstirring under argon atmosphere, and a solution of 4-picoline (in THF)carefully added to the cooled flask under constant stirring. After onehour, a solution of N-methoxy-N-methylbenzamide (in THF) was added overa period of 30 min. Formation of the product was monitored by HPLC andTLC. The reaction mixture was warmed to ambient temperature, duringwhich time the color changed from light yellow to orange, and thereaction quenched with crushed ice addition. THF was removed in vacuo,the reaction mixture was treated with saturated sodium bicarbonatesolution, and repeat extraction with ethyl acetate was done in aseparatory funnel. The combined organic extracts were treated withbrine, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure to yield an oily mixture. The crude mixture waspurified by column chromatography on silica gel (200-400 mesh) withproduct elution using ethyl acetate: hexane (1:4 v/v). The product,ketone 2-1, was obtained as a bright yellow solid in 69% (gravimetric)yield, 90% purity by HPLC and a mass (ESI) of m/z (MeOH)=198.18 (MH⁺).

Ethyl 4-oxo-4-phenyl-3-(pyridin-4-yl)butanoate (2-2): Sodium hydride andchilled anhydrous THF (100 mL) were combined in a round bottom flaskunder argon at 0° C. Compound 2-1 in anhydrous THF was added dropwiseover 1.5 h with constant stirring. After an additional 0.5 h, ethylbromoacetate in anhydrous THF was added dropwise, the reaction mixturestirred at ambient temperature until completion as monitored by HPLC.The reaction mixture color changed from pale yellow to bright yellow.The reaction was quenched by addition of crushed ice, THF removed invacuo, the reaction mix decanted into a saturated sodium bicarbonatesolution, and the resulting solution subjected to repeat extraction withethyl acetate using a separatory funnel. The combined organic extractswere treated with brine, dried over anhydrous sodium sulfate, andconcentrated under reduced pressure to yield an oily residue. Product2-2 was obtained by silica gel (200-400 mesh) column chromatographyusing ethyl acetate: methanol (3:1 v/v) elution. Product 2-2 wasobtained as a beige solid in 74% (gravimetric) yield, 90% purity byHPLC, and a mass (ESI) of m/z (MeOH)=284.75 (MH⁺).

6-Phenyl-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one (2-3): Compound2-2 was mixed with ethanol (400 mL) in a round bottom flask, hydrazineadded, and the resulting solution was heated (90° C.) under reflux withcontinuous stirring until completion (˜20 h) as monitored by HPLC. Thereaction mixture was cooled to ambient temperature and solvent removedunder reduced pressure. The residue was treated with ethyl acetatefollowed by ether addition, and the solution taken to dryness by rotaryevaporation. The white foam-like solid contained compound 2-3, with amass (ESI) of m/z (MeOH)=252.1 (MH⁺), was used for the next step withoutfurther purification.

6-Phenyl-5-(pyridin-4-yl)pyridazin-3-ol (2-4): Bromine in acetic acidwas added dropwise under continuous stirring to a round bottom flaskcontaining compound 2-3, the mixture refluxed at 70° C. until startingmaterial was no longer detectable (˜4 h) as monitored by HPLC. Themixture was cooled to ambient temperature, poured over crushed ice,neutralization with a sodium bicarbonate solution, and subjected torepeat extraction with ethyl acetate. The combined organic layerstreated with brine, dried over sodium sulfate and evaporated underreduced pressure to give a deep brown solid. Compound 2-4 was purifiedby silica gel (200-400 mesh) column chromatography using elution withethyl acetate: methanol (9:1 v/v). Compound 2-4 was obtained as a lightbrown solid in 68% (gravimetric) yield, 90% purity by HPLC, and a mass(ESI) m/z (MeOH)=250.20 (MW).

6-Chloro-3-phenyl-4-(pyridin-4-yl)pyridazine (2-5): Compound 2-5 wasobtained following a published protocol (Bioorg. Med. Chem. Lett. 2007,17, 414; herein incorporated by reference in its entirety). Compound 2-4was suspended in acetonitrile, POCl₃ (Reagent Plus Grade, 99%) added,the mixture heated to 90° C. for 3 h, cooled to ambient temperature,volatiles were removed in vacuo, the residual suspension poured intocrushed ice, stirred for 3 h at ambient temperature, and neutralizedwith 2.5N NaOH. The fine precipitate was subjected to repeat extractionwith ethyl acetate, the combined organic phases subjected to drying overanhydrous magnesium sulfate and concentration in vacuo. Product 2-5 waspurified from by silica gel (200-400 mesh) column chromatography usingethyl acetate:1% methanol. Product 2-5 was obtained as a white powder in82% (gravimetric) yield, with an HPLC purity of 98% and a mass (ESI) m/z(MeOH)=268.00 (MH⁺).

Production of kinase inhibitors from common intermediate (2-5). Thevarious kinase inhibitors were synthesized by reaction of a given aminewith compound 2-5 using a published protocol (Bioorg. Med. Chem. Lett.2007, 17, 414; herein incorporated by reference in its entirety).Compound 2-5 and 1-butanol were combined in a round bottom flask withthe respective amine, heated to 110° C. for approximately 15 h, cooledto ambient temperature, treated with water, and the aqueous layersubjected to repeat extraction with dichloromethane. The combinedorganic layers were subjected to drying with anhydrous sodium sulfateand concentration in vacuo. The final products were purified by silicagel column chromatography using volatile solvents for elution and finalprocessing.

6-(4-methylpiperazin-1-yl)-3-phenyl-4-(pyridin-4-yl)pyridazine(3=MW-066): Compound 2-5 was reacted with 1-methylpiperazine and takenthrough the protocol above to give product 3 as a beige powder in 89%(gravimetric) yield; MP: 190.2-190.7° C. (uncorrected); ¹H-NMR (500 MHz,CDCl₃): δ 8.57 (dd, J=1.55, 4.5 Hz, 2H); 7.34-7.24 (m, 5H); 7.12 (dd,J=1.6, 4.2 Hz, 2H); 6.84 (s, 1H); 3.81 (t, J=4.5, 4.6 Hz, 4H); 2.60 (s,4H); 2.39 (s, 3H); HPLC (tr/purity): 9.3 min>97% (HPLC method A); ESIm/z (MeOH): 332.1 (MH⁺); HRMS 331.1783 (calculated for C₂₀H₂₂N₅331.1797).

N, N-diethyl-6-phenyl-5-(pyridin-4-yl)pyridazin-3-amine (4=MW-177):Compound 2-5 was reacted with 10 equivalents of diethylamine andcompound 4 obtained by chromatographic elution with ethyl acetate:hexane (2:3 v/v) followed by crystallization in ethyl acetate and hexaneto give beige crystals in 70% overall yield (gravimetric). MP:121.5-122° C. (uncorrected). ¹H-NMR (500 MHz, CDCl₃): δ 8.57 (d, J=5.75Hz, 2H); 7.34-7.23 (m, 5H); 7.14 (dd, J=1.55, 4.45 Hz, 2H); 6.64 (s,1H); 3.71 (dd, J=7.1, 7.1 Hz, 4H); 1.29 (t, J=7.1, 9 Hz, 6H); HPLC(tr/purity): 12.4 min, >96% (HPLC method A); ESI m/z (MeOH): 305.10(MH⁺); HRMS 304.1682 (calculated for C₁₉H₂₀N₄ 304.1688).

N-methyl-6-phenyl-N-propyl-5-(pyridin-4-yl)pyridazin-3-amine (5=MW-207):Compound 2-5 was reacted with N-methylpropane-1-amine, processed asabove, and the final product obtained via chromatography using ethylacetate: hexane (1:1 v/v) as solvent, followed by crystallization inethyl acetate and hexane, to give the desired product 5 as whitecrystals in 74% (gravimetric) overall yield. MP: 160.5-161° C.(uncorrected). ¹H-NMR (500 MHz, CD₃OD): δ 8.47 (dd, J=1.6, 4.55 Hz, 2H);7.32-7.25 (m, 7H); 7.08 (s, 1H); 3.69 (t, J=7.4, 7.4 Hz, 2H); 3.22 (s,3H); 1.74 (m, 2H); 0.99 (t, J=7, 7.45 Hz, 3H); HPLC (tr/purity): 12.8min, >96% (HPLC method A); ESI m/z (MeOH): 305.10 (MH⁺); HRMS 304.1699(calculated for C₁₉H₂₀N₄ 304.1688).

N, N-dimethyl-6-phenyl-5-(pyridin-4-yl)pyridazin-3-amine (6=MW-105):Compound 2-5 was reacted with six equivalents of 40% dimethylamine at120° C. for 8 h and processed as above. The final product was obtainedvia crystallization from ethyl acetate and methanol to give the desiredproduct 6 as a light yellow crystalline solid in 90% yield(gravimetric). MP: 155.5-156° C. (uncorrected). ¹H-NMR (500 MHz, CDCl₃):δ 8.57 (d, J=5.8 Hz, 2H); 7.34-7.24 (m, 5H); 7.14 (dd, J=1.5, 4.65, 2H);6.72 (s, 1H); 3.27 (s, 6H); HPLC (tr/purity): 10.3 min>98% (HPLC methodA); ESI m/z (MeOH): 277.14 (MH⁺); HRMS 276.1384 (calculated for C₁₇H₁₆N₄276.1375).

Production of hydrochloride hydrate forms of 3, 4, 5 and 6. This wasdone following the protocol of Hu et al. (Bioorg. Med. Chem. Lett. 2007,17, 414; herein incorporated by reference in its entirety). Under theseconditions for the studies described here, elemental analyses of eachproduct indicated a mole ratio of HCl:compound of ˜2.

MW-066 (3) hydrochloride hydrate: EA calculated for C₂₀H₂₃Cl₂N₅: C,59.41; H, 5.73; Cl, 17.54; N, 17.32; experimentally found C, 59.08; H,5.69; Cl, 17.14; N, 17.11; O, 1.03.

MW-177 (4) hydrochloride hydrate: EA calculated for C₁₉H₂₆Cl₂N₄O₂: C,55.21; H, 6.34; Cl, 17.15; N, 13.55; O, 7.74; experimentally found C,56.30; H, 5.85; Cl, 20.23; N, 13.77; O, 3.32.

MW-207 (5) hydrochloride hydrate: EA calculated for C₁₉H₂₆Cl₂N₄O₂: C,55.21; H, 6.34; Cl, 17.15; N, 13.55; O, 7.74; experimentally found C,55.49; H, 6.17; Cl, 17.92; N, 13.60; O, 3.58.

MW-105 (6) hydrochloride hydrate: EA calculated for C₁₇H₂₄Cl₂N₄O₃: C,50.63; H, 6.00; Cl, 17.58; N, 13.89; O, 11.90; experimentally found: C,50.75; H, 5.89; Cl, 17.75; N, 13.90; O, 12.20.

Example 3. Synthesis of MW-077 (7) and SRM-075 (8)

N-methoxy-N-methyl-1-naphthamide (3-1): 1-naphthoyl chloride (1 eq), N,O-dimethyl-hydroxylamine hydrochloride (3 eq) and methylene chloride arecombined under argon, then cooled in an ice bath. Triethylamine (2.33eq) is added drop wise with stirring over 0.5 h. The contents of thereaction flask are then warmed to an ambient temperature slowly. After18 h, the reaction suspension is washed successively with 5% potassiumbisulfate, saturated sodium bicarbonate and brine. The organic layer isdried over anhydrous sodium sulfate then concentrated in vacuum to givecompound (3-1), yield 100%.

1-(naphthalen-1-yl)-2-(pyridin-4-yl)ethanone (3-2): To a stirringsolution of lithium diisopropylamide (1.5 eq) in THF at −78° C. asolution of 4-picoline (1 eq) in THF (10 ml) is added slowly. After 15min, a solution of N-Methoxy-N-methyl-1-naphthamide (1.2 eq) in THF (10ml) is added. After stirring for 5 minutes the reaction mixture isslowly allowed to warm to an ambient temperature. The formation of theproduct is monitored by HPLC. After the reaction is complete, thereaction mixture is poured into saturated aqueous sodium bicarbonate andextracted with ethyl acetate. The organic layers are washed with brine,dried over anhydrous magnesium sulphate and evaporated under reducedpressure to give the crude product. The crude product is then purifiedby flash column chromatography (25-100% EtOAc-hexane) OR triturationwith 10% ether/hexane gave the product (3-2), yield ˜69-70%.

Ethyl 4-(naphthalen-1-yl)-4-oxo-3-(pyridin-4-yl)butanoate (3-3): In anoven-dried 3-neck round bottom flask fitted with an internalthermometer, argon adapter and rubber septum, sodium hydride (1.1 eq)and anhydrous THF are combined under argon at 0° C. A solution ofcompound (3-2) (1 eq) in anhydrous THF is added drop wise over 0.5 husing an addition funnel. After stirring an additional 30 min, asolution of ethyl bromoacetate (1.5 eq) in anhydrous THF is addeddrop-wise. The reaction mixture stirred at an ambient temperature andmonitored using HPLC. After 18 h, when complete, the reaction mixture isslowly poured into water. Saturated sodium bicarbonate is added and theresulting solution is extracted several times with ethyl acetate. Thecombined organic extracts are dried over anhydrous sodium sulphate andthen concentrated under reduced pressure to give oil. Purification byflash column chromatography (hexane: ethyl acetate 70:30) gives theproduct (3-3), yield ˜20%.

6-(naphthalen-1-yl)-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one(3-4): The compound (3-3) (1 eq) and ethanol are combined and placed ina round bottom flask and to this hydrazine (2 eq) is added slowly. Theresulting solution is heated under reflux with continuous stirring untilthe reaction is complete as monitored by HPLC. Upon completion thereaction mixture is cooled to an ambient temperature and the solventevaporated in vacuo. Ethyl acetate is added followed by the addition ofether. The solution is again rotary evaporated to afford a foam-likesolid (3-4), which is used for the synthesis of next step withoutfurther purification, yield 100%.

6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-ol (3-5): Compound (3-4)is taken in a single-necked round bottom flask in acetic acid andbromine in acetic acid (1.5 eq) is added drop-wise to the solution usingan addition funnel. The reaction is refluxed at 70° C. and monitored byHPLC until all starting material consumed. The mixture is then cooled toan ambient temperature and poured into crushed ice to quench thereaction. The pH of the aqueous reaction mixture is adjusted to pH 7with 0.2N NaOH and then extracted with dichloromethane. The organiclayers are combined and purified by flash column chromatography oversilica gel column and eluting with dichloromethane and MeOH (5%) toafforded compound (3-5), yield 51%.

6-chloro-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (3-6): In around bottom flask compound 3-5 (1 eq) taken in ACN and POCl₃ (10 eq)was added slowly. The reaction mixture was stirred at 90° C., andmonitored by HPLC. When the starting materials disappeared, bring it toan ambient temperature and poured into crushed ice. The pH of themixture adjusted to pH 7 with 10N NaOH. The resulting mixture is thenexhaustively extracted with ethyl acetate and dichloromethane. Theorganic extracts are combined and dried over anhydrous magnesiumsulfate. The organic solvent evaporated in vacuo and the productpurified by flash column chromatography (Hexane: ethyl acetate) affordedcompound (3-6), yield 76%.

6-cyclopropyl-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (7=MW-077):Compound 3-6 (2 g, 6.2 mmol, 1 eq) was suspended in THF with 1.4 equivof cyclopropylboronic acid (760 mg, 8.8 mmol, 1.4 eq) in a heavy wallpressure vessel (Chemglass, Vineland, N.J.) and the reaction mixture waspurged with argon for 15 min. To this added 0.1 equiv of Pd (dppf)Cl₂CH₂Cl₂ (565 mg, 0.62 mmol), 2.5 equiv of silver oxide (3.65 g, 15.7mmol) and 3 equiv of potassium carbonate (2.6 g, 18.8 mmol). The mixturewas purged with argon and heated at 100° C. for 26 h in a sealed tube.The reaction mixture was cooled to ambient temperature and quenched witheither hydrogen peroxide (33%) or sodium hydroxide (10%). The aqueouslayer was extracted several times with ether and the ethereal layerswere combined dried over anhydrous sodium sulfate and concentrated invacuo resulting in a black mass. The crude mixture was purified bycolumn chromatography on silica gel (200-400 mesh) and eluted with ethylacetate: hexane (2:3 v/v) followed by crystallization with ethyl acetateand hexane to obtained SRM-077 (7) as a white crystals (0.75 g) 36.6%yield (gravimetric). MP 199.5-200 (uncorrected): ¹H-NMR (500 MHz,CDCl₃): δ 8.36 (dd, J=1.5, 4.5 Hz, 2H); 7.88-7.84 (m, 2H); 7.57 (d,J=8.3 Hz, 1H); 7.45-7.29 (m, 5H); 6.98 (dd, J=1.6, 4.5 Hz, 2H);2.36-2.30 (m, 1H); 1.42-1.35 (m, 2H); 1.31-1.24 (m, 2H). HPLC(tr/purity): 17.26 min/>97%. HRMS calculated for C₂₂H₁₇N₃ 323.1422;found 323.1423.

6-cyclopropyl-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazinehydrochloride hydrate: (8=SRM-075): In a round bottom flask fitted withcondenser and dry tube, compound 7 (SRM-077) (0.920 mg, 2.8 mmol) wassuspended in (˜20 mL) anhydrous isopropanol (99.5%, Aldrich) and heatedto 85° C. with stirring until dissolved. To the resulting solution 3equiv (0.725 μL) of ultrapure HCl (12N, JT Baker Ultrex® II, Product6900-05) was added at once inducing formation of solids in suspension.The resulting solution was stirred at 80° C. for 10 min, cooled toambient temperature and placed on ice for 2.5 h. The suspension was thentransferred to 4° C. for an additional 16 hr. The resulting yellowprecipitate was filtered on a medium frit sintered glass funnel usinghouse vacuum, immediately washed with (3×5 mL) of ice-cold anhydrousisopropanol followed by (3×5) of ice-cold anhydrous ether and air driedby house vacuum for 7 h. The precipitate was then dried in a glassdesiccator containing silica gel in vacuo until the compound gave aconstant weight. The final product (8=SRM-075) was then transferredusing a sterile/pyrogen-free spatula to a sterile storage vial under aClass II, laminar flow containment hood. Compound 8 (SRM-075) was ayellow powder (1.03 g) in 86% yield (gravimetric) compared to startingmaterial. ¹H NMR (500 MHz, CD₃OD): Δ 8.70 (t, J=1.5, 5.6 Hz, 2H), 8.40(s, 1H), 8.10 (d, J=8.2 Hz, 1H), 7.96-7.94 (m, 3H), 7.69-7.45 (m, 5H),2.67-2.61 (m, 1H), 1.61-1.51 (m, 4H). Hydrochloride hydrate formationwas confirmed by elemental analysis. Elemental analysis indicated aratio of ˜2. EA calculated for C₂₂H₂₃C₂N₃O₂: C, 61.12; H, 5.36; N, 9.72;Cl, 16.40; O, 7.40. Found C, 59.54; H, 5.38; N, 9.38; Cl, 15.49; O,9.46; Pd, 1.4 ppm. MP: 201-210° C. (decomposed) (uncorrected).

Example 3-1

Compound 46(6-(1-methylpiperidin-4-yl)-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine)(also described herein as SRM-137C) can be prepared in a similar manneras described in Example 3 by using (1-methylpiperidin-4-yl)boronic acidinstead of cyclopropylboronic acid.

Example 4. Synthesis of MW-125 (9)

2-(pyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one (4-2): A solution of4-picoline (0.11 g, 1.1 mmol, 0.11 mL) in THF (7 mL) under nitrogenatmosphere at −78° C. was treated with LDA [freshly prepared by treatingdiisopropylamine (0.12 g, 1.2 mmol, 0.17 mL) in THF (3 mL) with 2.5 Mn-BuLi (0.50 mL, 1.25 mmol) in hexanes for 30 minutes under nitrogenatmosphere in an ice bath] for 10 minutes. The mixture stirred 60minutes and was treated with neat N,O-dimethyl-2-naphthalenehydroxamicacid (4-1, 0.20 g, 0.93 mmol) dropwise over ten minutes. The mixturestirred an additional two hours then saturated aqueous ammonium chloride(2 mL) was added to the mixture and stirring continued an additional twohours while the temperature of the mixture rose to 20° C. The reactionmixture was diluted with ethyl acetate (50 mL) and washed with water(3×50 mL), brine (25 mL), dried (Na₂SO₄) and evaporated. The product waschromatographed on silica gel eluted with a gradient of ethyl acetate inhexanes (1:1 to 1:2 to 1:3) to leave the purified product 4-2 as a creamcolored crystalline solid (153 mg, 67%). ¹H-NMR (300 MHz, CDCl₃) δ 8.58(dd, J=4.4 Hz, J=1.8 Hz, 2H), 8.53 (s, 1H), 8.05 (dd, J=8.5 Hz, J=1.8Hz, 1H), 8.00-7.84 (m, 3H), 7.66-7.55 (m, 2H), 7.25 (m, 2H), 4.43 (s,2H); ESI MS (M+H)⁺=248; HPLC method A R_(t)=3.37 minutes.

Ethyl 3-(pyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate (4-3): To asolution of 2-(pyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one (4-2, 0.15g, 0.61 mmol) in 1,4-dioxane (6 mL) under nitrogen atmosphere at 20° C.was added sodium hydride (60% in mineral oil, 29 mg, 0.73 mmol) and themixture stirred for an hour. Ethyl bromoacetate (0.12 g, 0.73 mmol, 81μL) was added all at once and the mixture stirred for an hour. Saturatedaqueous ammonium chloride (2 mL) was added and stirring continued for anhour. The mixture was diluted with ethyl acetate (30 mL), washed withwater (2×30 mL), brine (20 mL), dried (Na₂SO₄) and evaporated to an oilyresidue that was purified by silica gel chromatography eluted with agradient of ethyl acetate in hexanes (1:1 to 2:1) to leave the productas a light yellow viscous oil (121 mg, 60%). ¹H-NMR (300 MHz, CDCl₃) δ8.53 (dd, J=4.4 Hz, J=1.5 Hz, 2H), 8.49 (s, 1H), 7.99 (dd, J=8.5 Hz,J=1.7 Hz, 1H), 7.92 (d, J=7.9 Hz, 1H), 7.87-7.82 (m, 2H), 7.62-7.50 (m,2H), 7.29 (dd, J=4.4 Hz, J=1.5 Hz, 2H), 5.27 (m, 1H), 4.12 (q, J=7.0 Hz,2H), 3.42 (dd, J=17.0 Hz, J=9.4 Hz, 1H), 2.78 (dd, J=17.0 Hz, J=5.6 Hz,1H), 1.21 (t, J=7.0 Hz, 3H); ESI MS (M+H)⁺=334; HPLC method A R_(t)=3.89minutes.

6-(naphthalen-2-yl)-5-(pyridin-4-yl)-2,3,4,5-tetrahydropyridazin-3-one(4-4): A solution of Ethyl3-(pyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate (4-3, 0.12 g, 0.36mmol) in ethanol (3 mL) was treated with hydrazine hydrate (0.25 g, 5mmol, 0.25 mL) and refluxed for 20 hours. The mixture was cooled to 20°C. and purified by reversed phase HPLC (method B). The product fractionswere treated with saturated sodium bicarbonate solution (50 mL) andextracted with ethyl acetate (2×50 mL). The combined extracts werewashed with brine (50 mL), dried (Na₂SO₄) and evaporated to leave 48 mg(44%) of beige solid. ¹H-NMR (300 MHz, CDCl₃) δ 8.76 (bs, 1H), 8.57 (d,J=5.3 Hz, 2H), 7.98 (dd, J=8.5 Hz, J=1.8 Hz, 1H), 7.95 (s, 1H), 7.85 (d,J=8.5 Hz, 1H), 7.86-7.76 (m, 2H), 7.55-7.46 (m, 2H), 7.21 (dd, J=4.4 Hz,J=1.2 Hz, 2H), 4.68 (d, J=6.5 Hz, 1H), 3.10 (dd, J=17.0 Hz, J=7.7 Hz,1H), 2.90 (dd, J=17.0 Hz, J=1.8 Hz, 1H); ESI MS (M+H)⁺=302; HPLC methodA R_(t)=3.11 minutes.

6-(Naphthalen-2-yl)-5-(pyridin-4-yl)-2,3-dihydropyridazin-3-one (4-5): Asolution of6-(naphthalen-2-yl)-5-(pyridin-4-yl)-2,3,4,5-tetrahydropyridazin-3-one(4-4, 25 mg, 0.083 mmol) in DMSO (0.5 mL) and water (0.02 mL) wastreated with N-bromosuccinimide (75 mg, 0.42 mmol). After a slightexotherm the solution was stirred 72 hours then purified directly byreversed phase HPLC (method B). The combined product fractions weretreated with saturated sodium bicarbonate solution (50 mL) and extractedwith ethyl acetate (2×25 mL). The combined extracts were washed withbrine, dried (Na₂SO₄) and evaporated to leave a beige solid (17 mg,69%). ¹H-NMR (300 MHz, CDCl₃) δ 11.13 (bs, 1H), 8.55 (bs, 2H), 7.80 (dd,J=9.1 Hz, J=2.1 Hz, 1H), 7.76-7.68 (m, 3H), 7.55-7.45 (m, 2H), 7.16 (dd,J=8.8 Hz, J=2.0 Hz, 1H), 7.09 (d, J=5.9 Hz, 2H), 7.05 (s, 1H); ESI MS(M+H)⁺=300; HPLC method A R_(t)=2.88 minutes.

6-Bromo-4-(pyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (4-6):6-(Naphthalen-2-yl)-5-(pyridin-4-yl)-2,3-dihydropyridazin-3-one (4-5,0.15 g, 0.50 mmol) was added to a solution of phosphorousoxybromide(0.62 g, 2.0 mmol) in acetonitrile (3 mL) and heated to reflux whilestirring for ten hours. The reaction mixture was cooled in an ice bath,treated with ice (2 g) and stirred for an hour. The mixture wasneutralized with solid sodium carbonate to a pH=10 followed by dilutionwith ethyl acetate (20 mL) and water (20 mL). The organic layer wasseparated, washed with brine (50 mL), dried (Na₂SO₄) and evaporated. Theproduct was purified by chromatography on silica gel eluted with ethylacetate in hexanes (3:1). The purified product was isolated as acolorless foamy solid (0.15 g, 83%). ¹H-NMR (300 MHz, CDCl₃) δ 8.59 (dd,J=4.4 Hz, J=1.8 Hz, 1H), 8.04 (d, J=1.2 Hz, 1H), 7.86-7.73 (m, 3H), 7.72(s, 1H), 7.58-7.47 (m, 2H), 7.33 (dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.15 (dd,J=4.4 Hz, J=1.7 Hz, 2H); ESI MS (M+H)₊=362, 364; HPLC method AR_(t)=3.71 minutes.

6-Cyclopropyl-4-(pyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (9): Amixture of 6-bromo-4-(pyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (4-6,40 mg, 0.11 mmol), cyclopropyl boronic acid (13 mg, 0.15 mmol),PdCl₂(dppf)·CH₂Cl₂ (8 mg, 0.011 mmol), silver oxide (64 mg, 0.28 mmol),potassium carbonate (46 mg, 0.33 mmol) and 1,4-dioxane (1 mL) wasstirred and purged with nitrogen. The mixture was heated to 80° C. for18 hours. The reaction mixture was cooled to 20° C. and filtered throughcelite (ethyl acetate wash). The filtrate was evaporated to dryness andthe crude product was purified by RPHPLC (method B). The productfraction was diluted with saturated sodium bicarbonate solution (50 mL)and the product was extracted with ethyl acetate (2×25 mL). The combinedorganic layers were washed with brine (2×25 mL), dried (Na₂SO₄) andevaporated to leave the product as a white solid (16 mg, 45%). ¹H-NMR(300 MHz, CD₃OD): δ 8.46 (d, J=4.4 Hz, 2H), 7.93 (d, J=1.5 Hz, 1H),7.88-7.77 (m, 4H), 7.65 (s, 1H), 7.56-7.46 (m, 2H), 7.37 (dd, J=8.5 Hz,J=1.7 Hz, 1H), 7.33 (dd, J=4.7 Hz, J=1.5 Hz, 1H), 2.4-2.35 (m, 1H),1.30-1.22 (m, 4H); ESI MS (M+H)⁺=324; HPLC method A R_(t)=3.47 minutes.

Example 5. Synthesis of MW-167 (10)

2-(3-fluoropyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one (5-3): Asolution of 3-fluoro-4-picoline (5-2, 3.2 g, 29 mmol, 2.9 mL) in THF(200 mL) under nitrogen atmosphere at −78° C. was treated with LDA[freshly prepared by treating diisopropylamine (5.3 g, 53 mmol, 7.4 mL)in THF (50 mL) with 2.5 M n-BuLi in hexanes for 30 minutes undernitrogen atmosphere in an ice bath] for 10 minutes. The mixture stirred60 minutes and was treated with neatN,O-dimethyl-2-naphthalenehydroxamic acid (5-1, 7.5 g, 35 mmol) dropwiseover ten minutes. The mixture stirred an additional two hours thensaturated aqueous ammonium chloride (20 mL) was added to the mixture andstirring continued an additional two hours while the temperature of themixture rose to 20° C. Approximately 200 mL of solvent was removed fromthe mixture in vacuo and the residue was diluted with ethyl acetate (200mL) and washed with water (3×100 mL). The product was extracted with INHCl (6×75 mL) and the combined acidic extracts were neutralized withsolid sodium carbonate to pH=9. The product was extracted with ethylacetate (2×100 mL) and the extracts were dried (Na₂SO₄) and evaporated.The product 5-3 was obtained as a light yellow crystalline solid (4.4 g,57%). ¹H-NMR (300 MHz, CDCl₃): δ 8.57 (s, 1H), 8.50 (d, J=1.5 Hz, 1H),8.41 (dd, J=4.6 Hz, J=1.0 Hz, 1H), 8.07 (dd, J=8.2 Hz, J=1.5 Hz), 8.01(d, J=18.7 Hz, 1H), 7.94 (m, 2H), 7.68-7.57 (m, 3H), 4.52 (s, 2H); ESIMS (M+H)⁺=266; HPLC method A R_(t)=4.29 minutes.

Ethyl 3-(3-fluoropyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate (5-4):To a solution of 2(3-fluoropyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one(5-3, 4.4 g, 17 mmol) in 1,4-dioxane (150 mL) under nitrogen atmosphereat 20° C. was added sodium hydride (60% in mineral oil, 0.80 g, 20 mmol)and the mixture stirred for an hour. Ethyl bromoacetate (3.3 g, 20 mmol,2.2 mL) was added all at once and the mixture stirred for 18 hours.Saturated aqueous ammonium chloride (20 mL) was added and stirringcontinued for an hour. The mixture was evaporated in vacuo toapproximately 50 mL in volume and diluted with ethyl acetate (200 mL).The mixture was washed with water (2×100 mL), brine (50 mL), dried(Na₂SO₄) and evaporated to an oily residue that was purified by silicagel chromatography eluted with a gradient of hexanes/ethyl acetate (3:1to 2:1) to leave the product as a light yellow viscous oil (4.4 g, 74%).¹H-NMR (300 MHz, CDCl₃): δ 8.54 (d, J=1.4 Hz, 1H), 8.46 (d, J=1.4 Hz,1H), 8.31 (dd, J=5.0 Hz, J=0.8 Hz, 1H), 8.00 (dd, J=8.5 Hz, J=1.6 Hz,1H), 9.94 (bd, J=8.2 Hz, 1H), 7.88-7.82 (m, 2H), 7.62-7.51 (m, 2H), 7.22(dd, J=5.0 Hz, J=5.0 Hz, 1H), 5.62 (dd, J=9.4 Hz, J=5.3 Hz, 1H), 4.13(q, J=7.0 Hz, 2H), 3.40 (dd, J=17.1 Hz, J=9.4 Hz, 1H), 2.80 (dd, J=17.1Hz, J=5.3 Hz, 1H), 1.21 (t, J=7.0 Hz); ESI MS (M+H)⁺=352; HPLC method AR_(t)=5.38 minutes.

6-(naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3,4,5-tetrahydropyridazin-3-one(5-5): A solution of Ethyl3-(3-fluoropyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate (5-4, 4.4 g,12.5 mmol) in ethanol (32 mL) and acetic acid (1.5 mL) was treated withhydrazine hydrate (12.5 g, 250 mmol, 12.5 mL) and refluxed for 20 hours.The mixture was cooled to 20° C., water (5 mL) was added and the beigeprecipitate was filtered, washed with 25% aqueous ethanol and air-driedto leave 1.85 g (46%). ¹H-NMR (300 MHz, CDCl₃): δ 8.92 (bs, 1H), 8.55(d, J=1.5 Hz, 1H), 8.30 (d, J=5.0 Hz, 1H), 7.99 (dd, J=8.5 Hz, J=1.7 Hz,1H), 7.90 (d, J=1.5 Hz, 1H), 7.88-7.78 (m, 3H), 7.55-7.45 (m, 2H), 7.04(dd, J=6.5 Hz, J=5.0 Hz, 1H), 5.02 (dd, J=7.9 Hz, J=1.5 Hz, 1H), 3.10(dd, J=17.3 Hz, J=7.9 Hz, 1H), 2.90 (dd, J=17.3 Hz, J=1.5 Hz, 1H); ESIMS (M+H)⁺=320; HPLC method A R_(t)=4.21 minutes.

6-(Naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3-dihydropyridazin-3-one(5-6): A solution of6-(naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3,4,5-tetrahydropyridazin-3-one(5-5, 3.2 g, 10 mmol) in DMSO (80 mL) and water (1.6 mL) was treatedwith N-bromosuccinimide (8.9 g, 50 mmol). After a slight exotherm thesolution was stirred 72 hours then diluted with water (300 mL) andextracted with ethyl acetate (3×150 mL). The combined organic layerswere washed with water (200 mL), saturated sodium bicarbonate solution(150 mL) and brine (50 mL). The solution was dried (Na₂SO₄) andevaporated. The solid residue was stirred rapidly in saturated sodiumbicarbonate solution (50 mL) and water (100 mL) for 18 hours thenfiltered, washed with water (5×25 mL) and dried under vacuum to leavethe product as a tan powder (3.1 g, 97%). ¹H-NMR (300 MHz, CDCl₃): δ11.69 (bs, 1H), 8.42 (dd, J=5.0 Hz, J=5.0 Hz, 1H), 8.38 (d, J=0.9 Hz,1H), 7.80 (d, J=7.7 Hz, 1H), 7.74-7.67 (m, 3H), 7.55-7.44 (m, 2H), 7.26(dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.20-7.13 (m, 1H), 7.11 (s, 1H); ESI MS(M+H)⁺=318; HPLC method A R_(t)=4.07 minutes.

6-Bromo-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (5-7):6-(Naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3-dihydropyridazin-3-one(5-6, 3.0 g, 9.5 mmol) was added to a solution of phosphorousoxybromide(11 g, 38 mmol) in acetonitrile (100 mL) and heated to reflux whilestirring for ten hours. The reaction mixture was cooled in an ice bath,treated with ice (50 g) and stirred for an hour. Approximately half ofthe solvent was removed in vacuo. The product mixture was diluted withethyl acetate (100 mL) and saturated sodium bicarbonate solution (300mL) was cautiously added with stirring until it reached a pH=8. Theorganic layer was separated, washed with brine (50 mL), dried (Na₂SO₄)and evaporated. The product was purified by chromatography on silica geleluted with a gradient of 40% to 50% ethyl acetate in hexanes. Thepurified product was isolated as a colorless foamy solid (2.2 g, 61%).¹H-NMR (300 MHz, CDCl₃): δ 8.47 (d, J=1.2 Hz, 1H), 8.41 (dd, J=5.0 Hz,J=1.0 Hz, 1H), 7.96 (d, J=1.5 Hz, 1H), 7.85-7.73 (m, 4H), 7.57-7.46 (m,2H), 7.41 (dd, J=8.8 Hz, J=2.1 Hz), 7.15 (dd, J=5.3 Hz, J=4.7 Hz, 1H);ESI MS (M+H)⁺=380, 382; HPLC method A R_(t)=5.29 minutes.

6-Cyclopropyl-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazine(10): A mixture of6-Bromo-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (5-7, 2.2g, 5.8 mmol), cyclopropyl boronic acid (0.70 g, 8.1 mmol),PdCl₂(dppf)·CH₂Cl₂ (0.42 g, 0.58 mmol), silver oxide (3.36 g, 14.5mmol), potassium carbonate (2.4 g, 17.4 mmol) and 1,4-dioxane (50 mL)was stirred and purged with nitrogen. The mixture was heated to 80° C.for 18 hours. The reaction mixture was cooled to 20° C. and filteredthrough celite (ethyl acetate wash). The filtrate was evaporated todryness and the crude product was purified by chromatography on silicagel eluted with 50% ethyl acetate in hexanes to leave the product as agum that crystallized upon trituration with hexane (1.1 g, 56%). ¹H-NMR(300 MHz, CDCl₃): δ 8.45 (d, J=1.4 Hz, 1H), 8.38 (dd, J=4.9 Hz, J=1.0Hz, 1H), 7.95 (m, 1H), 7.84-7.51 (m, 2H), 7.74 (s, 1H), 7.54-7.43 (m,3H), 7.33 (d, J=1.0 Hz, 1H), 7.14 (dd, J=5.6 Hz, J=5.0 Hz, 1H), 2.27 (m,1H), 1.38 (m, 2H), 1.24 (m, 2H); ESI MS (M+H)⁺=342; HPLC method AR_(t)=4.94 minutes.

6-Cyclopropyl-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazinehydrochloride (10·HCl): A sample of the cyclopropylpyridazine compound(10, 0.41 g, 1.2 mmol) was dissolved in methanol (20 mL) and treatedwith 1N HCl (1.4 mL, 1.4 mmol) and evaporated to dryness. The lightyellow amorphous solid was triturated in methylene chloride to leave alight yellow crystalline solid (0.41 g). ¹H-NMR (300 MHz, DMSO-d₆): δ8.51 (d, J=4.7 Hz, 1H), 8.47 (s, 1H), 7.91 (s, 1H), 7.90-7.79 (m, 3H),7.78 (s, 1H), 7.64 (dd, J=5.6 Hz, J=5.3 Hz, 1H), 7.58-7.46 (m, 2H), 7.42(d, J=8.2 Hz, 1H), 2.42 (m, 1H), 1.21 (narrow m, 4H).

Example 6: Synthesis of (11) MW-18-122, (12) MW-18-124, (13) MW-15-107,and (7) MW-16-077

N-methoxy-N-methyl-1-naphthamide (6-1): An ice cooled mixture of1-naphthoyl chloride (1 eq) and N, O-dimethyl-hydroxylaminehydrochloride (3 eq) in methylene chloride was treated dropwise withtriethylamine (2.33 eq) over 0.5 h under argon. The reaction mixture waswarmed slowly to an ambient temperature (20° C.) and stirred anadditional 18 h. The solvent was removed under reduced pressure, residuedissolved in ethyl acetate and washed successively with 5% potassiumbisulfate, saturated sodium bicarbonate and brine. The organic layer wassubjected to drying over anhydrous sodium sulfate and concentrated invacuo to leave the product 6-1 an oily mass, yield 100%.

1-(naphthalen-1-yl)-2-(pyridin-4-yl)ethanone (6-2): In a 2 Lround-bottom flask equipped with a stir bar, under N₂, was added4-methylpyridine (17.6 mL, 180 mmol) followed by anhydrous THF (500 mL).The mixture was then cooled down to −78° C. and LDA (90 mL, 2M in THF)was added dropwise over 1 h. After stirring for 2 h at −78° C. asolution of compound 6-1 (41 g, 190 mmol) in anhydrous THF (200 mL) wasadded to the reaction mixture drop wise over 30 min while stirring wascontinued for 1.5 h at −78° C. and 1.5 h at room temperature. Thereaction was then diluted with EtOAc and treated with saturated sodiumbicarbonate solution and repeat extraction with ethyl acetate was donein a separatory funnel. The combined organic extracts were treated withbrine, dried over anhydrous magnesium sulfate and concentrated underreduced pressure to yield an oily mixture. The crude mixture waspurified by column chromatography on silica gel (200-400 mesh) withproduct elution using ethyl acetate: hexane (1:4 v/v). The product,ketone 6-2, was obtained as a bright yellow solid (27.1 g,61%)(gravimetric) yield.

Ethyl 4-(naphthalen-1-yl)-4-oxo-3-(pyridin-4-yl)butanoate (6-3): In a 1L, round-bottom flask equipped with a stir bar, under N₂, was added 60%NaH (8.16 g, 204 mmol), followed by anhydrous DMSO (300 mL). Thereaction mixture was cooled down to 0° C. and stirred for 30 min. Thesolution of compound 6-2 (42 g, 170 mmol) in anhydrous DMSO (150 mL) wasadded via addition funnel at a steady drip over 45 min. Theheterogeneous solution was stirred for 30 min and then ethylbromoacetate (24.4 mL, 221 mmol) was added in one portion and the icebath removed. The reaction was stirred overnight, becoming homogenous.The resulting solution was poured into saturated ammonium chloride andextracted (EtOAc, 3×). The combined organic layers were washed withsaturated sodium bicarbonate, 1:1 H₂O: brine, followed by brine. Theresulting organic layer collected was dried over Na₂SO₄ and concentratedunder reduced pressure to yield an oily residue. Purification by flashchromatography gives compound 6-3 (11 g, 19%) (gravimetric) yield as asolid.

6-(naphthalen-1-yl)-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one(6-4): Compound 6-3 (11 g, 33 mmol) and ethanol (100 mL) were chargedinto a 350 mL sealed tube with a stir bar, under N₂. To this mixture wasadded hydrazine (2.1 mL, 66 mmol) and resulting solution stirred at 130°C. overnight. The reaction was concentrated under reduced pressure andthe crude 6-4 (9.4 g) as a yellow solid that was used for the next stepwithout further purification.

6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-ol (6-5): In a 500 mLround-bottom flask equipped with a stir bar was charged compound 6-4(9.4 g, 31.2 mmol) and glacial acetic acid (150 mL). The resultingsolution was heated at 95° C. for 50 min. To this mixture was added asolution of Br2 (1.8 mL, 34.3 mmol) in glacial acetic acid (10 mL). Thereaction was stirred for 1.5 h at 95° C. then concentrated in vacuo. Themixture was cooled to ambient temperature, was added EtOAc (200 mL) andH₂O (100 mL). The aqueous was adjusted to pH 8 by addition of 10% sodiumcarbonate and subjected to repeat extraction with ethyl acetate. Thecombined organic layers were washed with saturated sodium bicarbonatefollowed by brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. Compound 6-5 was purified by flash chromatographyyielding compound 6-5 (7 g), 75% (gravimetric) yield as off-white solid.

6-chloro-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (6-6): In a 500mL round-bottom flask equipped with a stir bar was charged with compound6-5 (7 g, 23.4 mmol), tetraethylbutyl ammonium chloride (5.4 g, 23.87mmol), diisopropylethylamine (4.12 mL, 23.6 mmol) and POCl₃ (103 g). Theresulting mixture was heated in an oil bath at 100° C. for 2 h thenconcentrated under reduced pressure. Remaining POCl₃ was azeotropicallyremoved using toluene. The crude was suspended in ethyl acetate and icedsaturated sodium bicarbonate was added. The aqueous layer was extractedseveral times with ethyl acetate and the combined organic layers werewashed with saturated sodium bicarbonate followed by brine. The organicphases subjected to drying over anhydrous sodium sulfate andconcentrated in vacuo and product was purified by silica gel (200-400mesh) column chromatography using ethyl acetate: hexane (2:3 v/v) aseluent. Product 6-6 was obtained as a beige powder (5.8 g) 78%(gravimetric) yield. ¹H-NMR (500 MHz, CDCl₃): δ 8.40 (d, J=5.9 Hz, 2H);7.93 (dd, J=8.15 Hz, 2H); 7.69 (d, J=3.75 Hz, 1H); 7.50-7.32 (m, 5H);6.99 (dd, J=1.6, 4.5 Hz, 2H). HPLC (tr/purity): 18.07 min, >96.8% (HPLCmethod A); mass (ESI) m/z (MeOH)=317.0 (MH⁺).

The above listed compounds (7, 11-13) were synthesized by reaction of agiven amine with compound 6-6 using the protocol described in Wattersonet al., PLOS ONE, 2013, 8, e66226). Compound 6-6 and 1-butanol werecombined in a round bottom flask with the respective amine, heated to110° C. for approximately 15 h, cooled to ambient temperature, treatedwith water, and the aqueous layer subjected to repeat extraction withdichloromethane. The combined organic layers were subjected to dryingwith anhydrous sodium sulfate and concentration in vacuo. The finalproducts were purified by silica gel column chromatography usingvolatile solvents for elution and final processing.

6-(4-methylpiperazin-1-yl)-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine(11): (MW-18): Compound6-chloro-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (6-6) (0.050 g,0.157 mmol) and 5 mL 1-butanol were combined in a heavy wall pressurevessel followed by the addition of 1-methylpiperazine (0.078 g, 0.79mmol). The pressure vessel was capped and heated at 120° C. for 16 h.The reaction mixture was then cooled to ambient temperature, transferredto a round bottom flask and concentrated in vacuo. The residue wastreated with 5 ml of Milli-Q water and the aqueous layer was extractedseveral times with dichloromethane using a separatory funnel. Thecombined organic layers were dried with anhydrous sodium sulfate andconcentrated in vacuo. The crude mixture was purified by columnchromatography on silica gel (200-400 mesh) and eluted with ethylacetate: hexane (3:1 v/v) to give the desired product 11 as a paleyellow powder (45 mg) in 79% yield (gravimetric). ¹H-NMR (500 MHz,CDCl₃): δ 8.35 (dd, J=5.0, 5.0 Hz, 2H); 7.85 (dd, J=5.0, 10 Hz, 2H);7.66 (dd, J=5.0, 7.5, 1H); 7.45-7.34 (m, 3H); 7.28 (dd, J=5.0, 7.5, 1H);6.97 (dd, J=5.0, 5.0, 3H); 3.90 (s, 4H); 2.70 (s, 4H); 2.46 (s, 3H);HPLC (tr/purity): 11.1 min, >98% (HPLC method A); (ESI) m/z (MeOH)=382.2(MH⁺). HRMS calculated for C₂₄H₂₃N₅ 381.19535, found 381.19575.

3-(naphthalen-1-yl)-6-(piperidin-1-yl)-4-(pyridin-4-yl)pyridazine (12):(MW-124): Compound6-chloro-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (6-6) (0.050 g,0.157 mmol) was reacted with piperidine (0.078 g, 0.79 mmol) in1-butanol, processed as above, and the final product obtained viachromatography using ethyl acetate: hexane (3:1 v/v) as eluent, to give12 as a beige powder (50 mg) in 89% (gravimetric) yield. ¹H NMR (500MHz, CDCl₃): δ 8.35 (dd, J=5.0, 5.0 Hz, 2H); 7.84-7.82 (m, 2H); 7.69 (d,J=10 Hz, 1H); 7.44-7.28 (m, 4H); 7.01 (t, J=5.0, 5.0 Hz, 2H); 6.96 (s,1H); 3.83 (t, J=5.0, 5.0 Hz, 4H); 1.77 (s, 6H). HPLC (tr/purity): 16.9min, >97% (HPLC method A); (ESI) m/z (MeOH)=367.2 (MH⁺).

N-cyclopropyl-6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-amine(13): (MW-107): Compound6-chloro-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (6-6) (0.600 g,1.8 mmol) was reacted with cyclopropanamine (0.645 g, 11.3 mmol) in1-butanol, processed as above, and the final product obtained via columnchromatography using ethyl acetate: hexane (4:1 v/v) as eluent, to give13 as a pale yellow crystals (360 mg) in 59% (gravimetric) yield. ¹H-NMR(500 MHz, CDCl₃: MeOD): δ 8.38 (dd, J=5.10 Hz, 2H), 7.85 (dd, J=5.10 Hz,2H); 7.64 (d, J=10 Hz, 1H); 7.45-7.34 (m, 3H), 7.28 (d, J=10 Hz, 1H),7.12 (s, 1H), 7.00-6.99 (m, 2H); 5.92 (s, br, 1H); 2.70-2.66 (m, 1H);0.94-0.90 (m, 2H); 0.74-0.71 (m, 2H). HPLC (tr/purity): 12.9 min, >96%(HPLC method A); ESI m/z (MeOH)=339.1 (MH⁺).

6-cyclopropyl-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (7):(MW-077): Compound6-chloro-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine (6-6) (2 g, 6.2mmol) was suspended in THF with 1.4 equiv of cyclopropylboronic acid(760 mg, 8.8 mmol in a heavy wall pressure vessel and the reactionmixture was purged with argon for 15 min. To this was added 0.1 equiv ofPd (dppf)Cl₂ CH₂Cl₂ (565 mg, 0.62 mmol), 2.5 equiv of silver oxide (3.65g, 15.7 mmol) and 3 equiv of potassium carbonate (2.6 g, 18.8 mmol). Themixture was purged with argon and heated at 100° C. for 26 h in a sealedtube. The reaction mixture was cooled to ambient temperature andquenched with either hydrogen peroxide (33%) or sodium hydroxide (10%).The aqueous layer subjected to repeat extraction with ether. Thecombined organic phases subjected to drying over anhydrous sodiumsulfate and concentrated in vacuo resulting in a black mass. The crudemixture was purified by silica gel (200-400 mesh) column chromatographyusing ethyl acetate: hexane (2:3 v/v) eluent, followed bycrystallization with ethyl acetate and hexane. Compound 7 was obtainedas a white crystalline solid in (1.1 g) 54% (gravimetric) yield; MP199.5-200° C. (uncorrected); ¹H NMR (500 MHz, CDCl₃): δ 8.37 (d, J=5 Hz,2H), 7.86 (m, 2H), 7.57 (d, J=8.3 Hz, 1H), 7.46-7.34 (m, 4H), 7.31 (dd,J=1.5, 4.6, 1H), 6.99 (dd, J=5 Hz, 2H), 2.35-2.32 (m, 1H), 1.41-1.39 (m,2H), 1.28-1.25 (m, 2H); HPLC (tr/purity): 17.26 min>97% (HPLC method A);ESI m/z (MeOH): 324.10 (MH⁺); HRMS 323.1423 (calculated for C₂₂H₁₇N₃323.1422).

Example 6-1

Compound 46(6-(1-methylpiperidin-4-yl)-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine)(also described herein as SRM-137C) can be prepared in a similar manneras described in Example 6 by using (1-methylpiperidin-4-yl)boronic acidinstead of cyclopropylboronic acid.

Example 7

N-methoxy-N-methyl-1-naphthamide (7-1): A mixture of 1-naphthoylchloride (5.0 g, 26 mmol) and N,O-dimethylhydroxylamine hydrochloride(2.8 g, 29 mmol) in dichloromethane (250 mL) was stirred and cooled inan external ice bath while it was treated with diisopropylethylamine(7.7 g, 60 mmol, 11 mL) dropwise over 20 minutes. The mixture rose to20° C. over 2 h and stirred an additional 18 h. The solvents wereevaporated in vacuo and the residue was taken up in ethyl acetate (200ml), washed with 1N HCl (100 mL), water (100 mL) and brine (50 mL). Theorganic layer was dried (Na₂SO₄) and evaporated to a tan oil (5.6 g,100%). ¹H NMR (300 MHz, CDCl₃): δ 7.95-7.85 (m, 3H), 7.58-7.45 (m, 4H),3.55 (bs, 3H), 3.40 (bs, 3H). LC/MS (method A) R_(t)=4.31 min,purity>95%, (M+H⁺)=216.

2-(3-fluoropyridin-4-yl)-1-(naphthalen-1-yl)ethan-1-one (7-3): A freshsolution of LDA was prepared by treating a solution of diisopropylamine(3.5 g, 35 mmol, 4.8 ml) in anhydrous THF (50 mL) under nitrogenatmosphere in an external ice bath with a solution of n-butyllithium(2.5 M in hexanes, 14 mL, 35 mmol) and allowing it to stir for 30minutes. The LDA solution was added via cannula to a solution of3-fluoro-4-picoline (7-2) (2.6 g, 23 mmol, 2.3 mL) in THF (100 ml) undernitrogen atmosphere at −78° C. over 20 minutes followed by additionalstirring for 60 minutes. A solution of N-methoxy-N-methyl-1-naphthamide(7-1) (5.5 g, 26 mmol) in THF (50 mL) was added dropwise to thefluoropicoline anion solution over 30 minutes ensuring the temperaturedid not rise above −70° C. The mixture warmed to 20° C. over 6 h thenstirred an additional 12 h. The mixture was treated with saturatedammonium chloride solution (20 mL) and stirred 30 minutes. The solventswere evaporated to 20% of the original volume (approx.) and the residuewas dissolved in ethyl acetate (200 mL) and washed with water (100 mL)and brine (50 mL). The organic layer was dried (Na₂SO₄) and evaporatedin vacuo. The crude product was chromatographed on silica gel elutedwith a gradient of ethyl acetate in hexanes (33%-50%) to leave theproduct as a light yellow solid (3.2 g, 53%). This product was dissolvedin ethyl acetate (50 mL) and extracted with 1N HCl (2×50 mL). The acidlayers were cautiously neutralized to pH=9 and extracted with ethylacetate (2×50 mL). The extracts were dried (Na₂SO₄) and evaporated toleave a beige solid (1.2 g, 17%). ¹H NMR (300 MHz, CDCl₃): δ 8.65 (d,J=8.5 Hz, 1H), 8.49 (bs, 1H), 8.40 (bs, 1H), 8.05 (d, J=6.8 Hz, 1H),8.02 (d, J=6.1 Hz, 1H), 7.89 (d, J=8.5 Hz, 1H), 7.63-7.53 (m, 3H), 7.29(dd, J=5.6 Hz, J=5.2 Hz), 4.46 (s, 2H). LC/MS (method A) R_(t)=4.32 min,purity>90%, (M+H⁺)=266.

Ethyl 3-(3-fluoropyridin-4-yl)-4-(naphthalen-1-yl)-4-oxobutanoate (7-4):A solution of 2-(3-fluoropyridin-4-yl)-1-(naphthalen-1-yl)ethan-1-one(7-3) 1.2 g, 4.5 mmol) in dioxane (25 mL) was treated with sodiumhydride (60% in mineral oil, 0.20 g, 5.1 mmol) under nitrogen atmosphereand stirred for 60 minutes. The resulting mixture was treated with ethyl2-bromoacetate (0.85 g, 5.1 mmol, 0.57 mL) and stirred 3 h. The mixturewas treated with saturated aqueous ammonium chloride (5 mL) and stirred30 minutes. The solvent volume was reduced to 20% of the original volume(approx.) and the residue was dissolved in ethyl acetate (50 mL) andsaturated aqueous sodium bicarbonate solution (50 mL). The organic layerwas separated, washed with brine (50 mL), dried (Na₂SO₄) and evaporatedin vacuo. The residue was dissolved in aqueous methanol (20 mL) andpurified by RPHPLC (method B). The product fractions were combined andevaporated to remove acetonitrile. The residue was treated withsaturated aqueous sodium bicarbonate solution and extracted with ethylacetate (2×25 mL). The combined extracts were washed with brine, dried(Na₂SO₄) and evaporated to leave the desired product as an oil (830 mg,53%). ¹H NMR (300 MHz, CDCl₃): δ 8.47 (d, J=8.2 Hz, 1H), 8.36 (s, 1H),8.30 (d, J=4.7 Hz, 1H), 8.01 (d, J=7.0 Hz, 1H), 7.96 (d, J=4.2 Hz, 1H),7.83 (d, J=7.4 Hz, 1H), 7.60-7.43 (m, 3H), 7.28 (dd, J=5.6 Hz, J=5.6Hz), 5.50 (dd, J=9.4 Hz, J=5.4 Hz, 1H), 4.18 (q, J=7.0 Hz, 2H), 3.47(dd, J=17.0 Hz, J=9.4 Hz, 1H), 2.77 (dd, J=17.0 Hz, J=5.4 Hz, 1H), 1.23(t, J=7.0 Hz, 3H). LC/MS (method A) R_(t)=5.38 min, purity>95%,(M+H⁺)=352.

5-(3-Fluoropyridin-4-yl)-6-(naphthalen-1-yl)-4,5-dihydropyridazin-3(2H)-one(7-5): A solution of ethyl3-(3-fluoropyridin-4-yl)-4-(naphthalen-1-yl)-4-oxobutanoate (0.82 g, 2.3mmol) in ethanol (95%, 10 mL) was treated with 85% hydrazine (4.5 mL)and acetic acid (0.30 mL) and the mixture was heated to reflux for 18 h.The solvents were evaporated and the residue was purified by RPHPLC(method B). The product fraction was treated with saturated sodiumbicarbonate solution and extracted with ethyl acetate (2×50 mL). Thecombined extracts were washed with brine, dried (Na₂SO₄) and evaporatedin vacuo to leave a tan solid (180 mg, 25%). ¹H NMR (300 MHz, CDCl₃): δ8.85 (bs, 1H), 8.39 (d, J=1.4 Hz, 1H), 8.32 (d, J=5.0 Hz, 1H), 8.29 (d,J=8.5 Hz, 1H), 7.86 (m, 2H), 7.61-7.51 (m, 2H), 7.41-7.31 (m, 2H), 7.18(dd, J=5.9 Hz, J=5.3 Hz, 1H), 4.79 (dd, J=7.9 Hz, J=4.4 Hz, 1H), 3.20(dd, J=17.2 Hz, J=7.9 Hz, 1H), 2.93 (dd, J=17.2 Hz, J=4.4 Hz, 1H). LC/MS(method A) R_(t)=3.89 min, purity>95%, (M+H⁺)=320.

5-(3-Fluoropyridin-4-yl)-6-(naphthalen-1-yl)pyridazin-3(2H)-one (7-6): Asolution of5-(3-fluoropyridin-4-yl)-6-(naphthalen-1-yl)-4,5-dihydropyridazin-3(2H)-one(170 mg, 0.53 mmol) in DMSO (3 mL) and water (85 μL) was treated withN-bromosuccinimide (470 mg, 2.7 mmol). The reaction became slightlyexothermic while stirring then cooled to 20° C. and stirred anadditional 18 h. The reaction mixture was diluted with ethyl acetate (50mL), washed with saturated sodium bicarbonate solution (50 mL) and brine(50 mL). The solution was dried (Na₂SO₄) and evaporated. The crudeproduct was purified by RPHPLC (method B) and the product fractions werecombined, treated with saturated sodium bicarbonate solution andextracted with ethyl acetate (2×25 mL). The combined extracts werewashed with brine (25 mL), dried (Na₂SO₄) and evaporated to leave theproduct as a white foamy solid (150 mg, 89%). ¹H NMR (300 MHz, CDCl₃): δ11.66 (bs, 1H), 8.26 (bs, 1H), 8.11 (d, J=5.0 Hz, 1H), 7.85-7.75 (m,2H), 7.70-7.60 (m, 1H), 7.50-7.27 (m, 4H), 7.14 (s, 1H), 6.86 (dd, J=5.9Hz, J=5.3 Hz, 1H). LC/MS (method A) R_(t)=3.92 min, purity>95%,(M+H⁺)=318.

6-chloro-4-(3-fluoropyridin-4-yl)-3-(naphthalen-1-yl)pyridazine (7-7):6-(Naphthalen-1-yl)-5-(3-fluoropyridin-4-yl)-2,3-dihydropyridazin-3-one(7-6, 0.120 g, 0.37 mmol) and 4 mL acetonitrile was taken in a flask,fitted with condenser and dry tube and added phosphorousoxychloride (145mg, 0.94 mmoL, 90 μL). Reflux at 90° C. for 5 hr (reaction was monitoredby HPLC). Reaction mixture was cooled at ambient temperature and thenremove POCl₃ in reduced pressure, added crushed ice and cautiously added10N NaOH with stirring until it reaches a pH ˜8. Fine precipitateobserved. Extract with dichloromethane several times and dried (Na₂SO₄)and evaporated. Product was purified over a small silica gel filtercolumn eluted with 40% ethyl acetate and Hexane. The purified compoundwas isolated as light pinkish powder (115 mg), 92% (gravimetric) yield.HPLC (tr/purity): 20.06 min, >95% (HPLC method A), mass (ESI) m/z=336.0(M+1).

4-(3-fluoropyridin-4-yl)-6-(4-methylpiperazin-1-yl)-3-(naphthalen-1-yl)pyridazine(14): (MW-109): A mixture of6-chloro-4-(3-fluoropyridin-4-yl)-3-(naphthalen-1-yl)pyridazine (7-7,0.090 g, 0.26 mmol), 5 mL ethanol and 1-methylpiperazine (0.13 g, 0.15mL, 1.3 mmol) was taken in a heavy glass vial and blanket with argon andcapped immediately. The reaction vial was heated at 120° C. for 26 hr.(reaction was monitored by HPLC). Reaction mixture was cooled to ambienttemperature and removes the volatiles under reduced pressure. To theyellow residue added 1 mL water and extracted several times withdichloromethane. The combined organic solvent was dried over anhydroussodium sulphate and evaporated under reduced pressure. The product 14was purified over silica gel (200-400 mesh) column with ethyl acetate:hexane (2:3 v/v) eluent. The purified product 14 was isolated as fluffybeige color solid (90 mg), 86% (gravimetric) yield. ¹H-NMR (500 MHz,CDCl₃): δ 8.29 (d, 1H); 8.06 (d, J=5 Hz, 1H); 7.80 (dd, J=5, 10 Hz, 2H);7.67 (d, J=10 Hz, 1H); 7.42-7.28 (m, 4H); 7.0 (s, 1H); 6.81 (t, J=5, 5Hz, 1H); 3.92 (bs, 4H); 2.74 (bs, 4H); 2.49 (s, 3H). HPLC (tr/purity):12.1 min, >97% (HPLC method A), mass (ESI) m/z=400.2. HRMS calculatedfor C₂₂H₂₂FN₅, 399.18592 found 399.18602.

Example 8

4-bromo-1,2-dihydropyridazine-3,6-dione (8-1): Hydrazine sulfate (2.25g, 17.2 mmol) was dissolved in boiling water (20 mL) with stirring. Tothis solution, bromomaleic anhydride (2.6 mL, 28.2 mmol) was added dropwise via addition funnel, the mixture heated (100° C.) under reflux for19 h, then cooled to ambient temperature. The resulting whiteprecipitate was filtered on a medium frit sintered glass funnel, washedwith acetone (3×5 mL), and air dried in vacuo to give4-bromo-1,2-dihydropyridazine-3,6-dione (2.85 g) as a white powder in87% yield (gravimetric) with a melting point of 262° C.

4-(pyridin-4-yl)-1,2-dihydropyridazine-3,6-dione (8-2): Essentially aspreviously described in Watterson et al., PLOS ONE, 2013, 8, e66226,compound 8-1 (2 g, 10.4 mmol, 1 eq) and pyridin-4-yl boronic acid (14.3mmol, 1.76 g, 1.37 eq) were suspended in dimethoxyethane and water (10:1v/v) in a heavy wall pressure vessel and purged with argon for 15 min.Tetrakis (triphenylphosphine) palladium (0.1 eq) and sodium carbonate (3eq) were added, the vessel immediately capped, the reaction mixtureheated (110° C.) for 18 h, then cooled to ambient temperature andsubjected to filtration on a medium frit sintered glass funnelcontaining Celite® 545. The filtrate was concentrated in vacuo and theconcentrate triturated with hexane. The yellow product 8-2 (2.2 g)exhibited a mass (ESI) of m/z (MeOH)=190.06 (MH⁺), and was taken to thenext step without further purification.

3,6-dichloro-4-(pyridin-4-yl)pyridazine (8-3): Essentially as describedin Watterson et al., PLOS ONE, 2013, 8, e66226, compound 8-2 (2.2 g,11.6 mmol) was suspended in 6.25 mL phosphorus oxychloride in acondenser-fitted round bottom flask, heated (90° C.) for 24 h, cooled toambient temperature and volatiles removed in vacuo. The dark residue waspoured onto crushed ice, stirred (2 h), and the mixture neutralized withsaturated sodium carbonate solution. The fine precipitate was subjectedto replicate extraction with dichloromethane in a separatory funnel, thecombined organic phases subjected to drying over anhydrous sodiumsulfate, concentrated in vacuo, and subjected to column chromatographyon silica gel (200-400 mesh) using a ethyl acetate:hexane (3:2 v/v)eluent. The desired product 8-3 exhibited 97% purity by HPLC and a mass(ESI) of m/z (MeOH)=225.99 (MH⁺). The overall yield (gravimetric) fromproduct 8-1 to 8-3 was approximately 36%.

6-chloro-N-methyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4a): Followingthe published protocol in Watterson et al., PLOS ONE, 2013, 8, e66226,compound 8-3 (0.750 g, 3.3 mmol, 1 eq) and 25 mL 1-butanol were placedin a heavy wall pressure vessel (Chemglass, Vineland, N.J.) and 40%methylamine (0.618 g, 19.9 mmol, 6 eq) was added, the vessel capped andheated at 110° C. for 4 h. The reaction mixture was cooled to ambienttemperature, transferred to a round bottom flask and concentrated invacuo. The residue was treated with 10 mL of water and the aqueous phasesubjected to replicate extraction with ethyl acetate using separatoryfunnel. The combined organic layers were dried with anhydrous sodiumsulfate and evaporated under reduced pressure leaving a yellow solid.The reaction mixture was purified by crystallization with methanol togive product 8-4a as a white crystals (0.500 g) in 68% (gravimetric)yield, HPLC (tr/purity): 10.13 min>95% (HPLC method A), and a mass (ESI)m/z (MeOH)=221.10 (MH⁺).

N-methyl-6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-amine (15):(MW-156): CompoundN-methyl-6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-amine (8-4a)(0.450 g, 2.02 mmol, 1 eq) and 1-napthylboronic acid (0.480 g, 2.79mmol, 1.37 eq) was suspended in dimethoxyethane (DME) and water mixture(10:1 v/v) in a heavy wall pressure vessel (Chemglass, Vineland, N.J.)and purged with argon for 15 min. Subsequently,Tetrakis(triphenylphosphine)palladium (0.212 g, 9 mol %,) and Na₂CO₃(0.670 g, 6.32 mmol, 3.1 eq) were added vessel purged with argon andimmediately capped with a Teflon bushing, heated (110° C.) for 16 hr,and cooled to ambient temperature. The reaction mixture was subjected tofiltration on a medium frit sintered glass funnel containing Celite®545. The resultant filtrate was concentrated in vacuo, the residuedissolved in ethyl acetate and subjected to water wash washed (3×10 mL)using a separatory funnel. The organic layer was dried over anhydroussodium sulfate and concentrated by rotary evaporation under reducedpressure. The crude mixture was subjected to silica gel (200-400 mesh)column chromatography with product elution using ethyl acetate: methanol(19:1 v/v). The product 15 was obtained as pale yellow crystals (464 mg)in 63% yield (gravimetric). ¹H-NMR (500 MHz, CDCl₃): δ 8.33 (dd, J=1.6,4.5 Hz, 2H); 7.83 (d, J=8.25, 2H); 7.65 (d, J=8.4, 1H); 7.43-7.26 (m,4H); 6.94 (dd, J=1.6, 4.5 Hz, 2H); 6.75 (s, 1H); 5.29 (m, 1H); 3.13 (d,J=4.95, 3H). HPLC (tr/purity): 11.60 min>97% (HPLC method A); mass (ESI)of m/z (MeOH): 313.10 (MH⁺); HRMS calculated for C₂₀H₁₆N₄ 312.1375;found 312.1378.

N-methyl-6-(naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3-amine (16):(MW-200): Product 16 was produced using the same protocol ‘Suzuki’reaction as above for 15 but using 2-napthylboronic acid (427 mg, 2.48mmol, 1.37 eq) was suspended in dimethoxyethane (DME) and water mixture(10:1 v/v). Product 16 was purified by silica gel (200-400 mesh) columnchromatography with product elution using ethyl acetate: hexane (1:1v/v). The final product 16 was obtained as white crystals (360 mg) in63% (gravimetric) yield. ¹H-NMR (500 MHz CDCl₃): δ 8.54 (dd, J=1.55,4.45 Hz, 2H); 7.91 (s, 1H); 7.80-7.68 (m, 3H); 7.49-7.31 (m, 3H); 7.14(dd, J=1.6, 4.45 Hz, 2H); 6.68 (s, 1H); 5.21 (b, 1H, —NH); 3.15 (d,J=4.95, 3H). HPLC (tr/purity): 12.15 min>97% (HPLC method A); mass (ESI)m/z (MeOH): 313.10 (MH⁺); HRMS calculated for C₂₀H₁₆N₄ 312.1335; found312.1375.

6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4b): Product8-4b was made following the same protocol described above for 8-4a,using 40% dimethylamine (0.590, 13.2 mmol, 6 eq) in ethanol and heated(110° C.) for 16 h. Product 8-4b was obtained as a yellow solid in 89%(gravimetric) yield, with an apparent HPLC (tr/purity): 11.45 min>98%(HPLC method A), ¹H NMR (500 MHz, CDCl₃): δ 8.76 (d, J=5.0 Hz, 2H); 7.41(m, 2H); 6.70 (s, 1H); 3.22 (s, 6H); and a mass (ESI) m/z (MeOH)=235.10(MH⁺).

6-(4-fluoronaphthalen-1-yl)-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine(17): MW-078: Product 17 was synthesized using the same ‘Suzuki’reaction protocol for 15 as above but using 8-4b. Compound6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4b) (1 g, 4.2mmol) and 4-fluoronaphthalen-1-yl)boronic acid (1.1 g, 5.85 mmol, 1.3eq) was suspended in dimethoxyethane (DME) and water mixture (10:1 v/v)and followed the above protocol. The final product 17 was purified bysilica gel (200-400 mesh) column chromatography using ethyl acetate:hexane (3:7 v/v) eluent. Product 17 was obtained as a crystalline powder(710 mg) in 51% (gravimetric) yield; ¹H NMR (500 MHz, CDCl₃): δ 8.37 (d,J=4.6 Hz, 2H); 8.10 (d, J=8.3 Hz, 1H); 7.69 (d, J=8.5 Hz, 1H); 7.50-7.39(m, 2H); 7.22-7.04 (m, 2H); 6.99 (d, J=5.8 Hz, 2H); 6.84 (s, 1H); 3.33(s, 6H). HPLC (tr/purity): 13.7 min>97% (method A); mass (ESI) m/z(MeOH): 345.10 (MH⁺); HRMS calculated for C₂₁H₁₇FN₄ 344.1437; found344.1442.

6-(7-fluoronaphthalen-1-yl)-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine(18): (MW-085): Product 18 was synthesized using the same protocol asabove for 15. Compound6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4b) and(7-fluoronaphthalen-1-yl) boronic acid was suspended in dimethoxyethane(DME) and water (10:1 v/v) and followed the same protocol above. Theproduct 18 was purified by silica gel (200-400 mesh) columnchromatography using ethyl acetate and hexane (2:3 v/v) eluent. Product18 was obtained as light yellow needle-like crystals (200 mg) in 62%(gravimetric) yield. ¹H NMR (500 MHz, CDCl₃): δ 8.37 (dd, J=1.6, 4.5 Hz,2H); 7.83 (m, 1H); 7.35-7.28 (m, 3H); 7.22-7.18 (m, 1H); 6.99 (dd,J=6.2, 4.5 Hz, 2H); 6.84 (s, 1H); 3.33 (s, 6H). LC/MS (tr/purity): 1.82min, 99.6%; mass (ESI) m/z (MeOH): 345.7 (MH⁺).

6-(2-methoxynaphthalen-1-yl)-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine(19): (MW-155): Product 19 was synthesized using the same protocol asabove for 15. Compound6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4b) (0.400 g,1.7 mmol) and (2-methoxynaphthalen-1-yl) boronic acid (0.334 g, 2.3mmol) was suspended in dimethoxyethane (DME) and water (10:1 v/v) andfollowed the same protocol above. The product 19 was purified by silicagel (200-400 mesh) column chromatography using (2:3 v/v) ethyl acetateand hexane followed by crystallization in methanol and hexane. Product19 was obtained as white crystals (398 mg) in 66% (gravimetric) yield.¹H NMR (500 MHz, CDCl₃): δ 8.34 (dd, J=5.5 Hz, 2H); 7.83 (d, J=10 Hz,1H); 7.78 (d, J=5 Hz, 1H); 7.64 (d, J=10 Hz, 1H); 7.40-7.31 (m, 2H);7.04 (d, J=5 Hz, 1H); 6.95 (dd, J=5, 5 Hz, 2H); 6.81 (s, 1H); 3.47 (s,3H); 3.30 (s, 6H). HPLC (tr/purity): 12.4 min>97% (method A); exhibiteda mass (ESI) m/z (MeOH): 357.10 (MW).

6-(isoquinolin-5-yl)-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine(20): (MW-082): Product 20 was synthesized using the same protocol asabove for 15. Compound6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4b) (0.500 g,2.1 mmol, 1 eq), and isoquinolin-5-ylboronic acid (0.504 g, 2.9 mmol,1.3 eq) was suspended in dimethoxyethane (DME) and water (10:1 v/v) andfollowed the same protocol above. The combined organic extracts weredried over anhydrous sodium sulfate, and concentrated under reducedpressure using rotary evaporator. The product 20 was purified by silicagel (200-400 mesh) column chromatography using ethyl acetate and 5%methanol eluent. Product 20 obtained as yellowish-white powder (470 mg)in 68% (gravimetric) yield. ¹H-NMR (500 MHz, CDCl₃): δ 9.25 (s, 1H);8.42-8.38 (m, 3H); 7.97 (d, J=8.0, 1H); 7.59-7.45 (m, 3H); 6.97 (dd,J=1.5, 4.5, 2H); 6.85 (s, 1H); 3.33 (s, 6H). HPLC (tr/purity): 9.2min>97% (method A); exhibited a mass (ESI) m/z (MeOH): 328.10 (MW).

6-(6-fluoroquinolin-8-yl)-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine(21): (MW-165): Product 21 was synthesized using the same protocol asabove for 15. Compound6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4b) (0.400 g,1.7 mmol, 1 eq), and (6-fluoroquinolin-8-yl)boronic acid (0.437 g, 2.2mmol, 1.3 eq) was suspended in dimethoxyethane (DME) and water (10:1v/v) and followed the same protocol above. The product 21 was purifiedby silica gel (200-400 mesh) column chromatography using ethyl acetateand hexane (4:1 v/v) eluent, followed by crystallization with ethylacetate. Product 21 obtained as white crystals (250 mg) in 45%(gravimetric) yield. ¹H-NMR (500 MHz, CDCl₃): δ 8.4 (dd, J=5.5 Hz, 1H);8.27 (dd, J=5.5 Hz, 2H); 7.96 (dd, J-=5.5 Hz, 1H); 7.90 (dd, J=5.5 Hz,1H); 7.44 (d, J=5, 7.5 Hz, 1H); 7.18 (dd, J=5, 10 Hz, 1H); 7.08 (d, J=5Hz, 2H); 6.80 (s, 1H); 3.30 (s, 6H); HPLC (tr/purity): 10.9 min>97%(method A); exhibited a mass (ESI) m/z (MeOH): 346.10 (MH⁻).

6-(1H-indol-5-yl)-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine (22):(MW-066): Product 22 was synthesized using the same protocol as abovefor 15. Compound 6-chloro-N,N-dimethyl-5-(pyridin-4-yl)pyridazin-3-amine(8-4b) (0.500 g, 2.1 mmol, 1 eq), (1H-indol-5-yl)boronic acid (0.470 g,2.9 mmol, 1.3 eq) was suspended in dimethoxyethane (DME) and water (10:1v/v) and followed the same protocol above The product 22 was purified bycolumn chromatography on silica gel (200-400 mesh) with product elutionusing ethyl acetate and methanol (5%) followed by crystallization withethyl acetate. The product 22 obtained as pale yellow powder (410 mg) in61% (gravimetric) yield. ¹H NMR (500 MHz CD₃OD): δ 8.42 ((dd, J=1.6, 4.6Hz, 2H); 7.51 (d, J=1.25 Hz, 2H); 7.29 (m, 4H); 7.15 (s, 1H); 6.95 (dd,J=1.6, 8.4 Hz, 1H); 6.39 (m, 1H); 3.25 (s, 6H). HPLC (tr/purity): 10.2min>97% (method A); a mass (ESI) of m/z (MeOH): 316.10 (MH⁺).

6-chloro-N, N-diethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4c): Product8-4c was made following the same protocol described above for 8-4a,using compound 8-3 (0.600 g, 2.65 mmol) and diethylamine (1.16 g, 15.9mmol, 6 eq) in 1-butanol and heated (150° C.) for 24 h. Product 8-4c wasobtained as a yellow solid (470 mg) in 72% (gravimetric) yield, with anapparent HPLC (tr/purity): 14.6 min>98% (HPLC method A), and a mass(ESI) m/z (MeOH)=263.10 (MH⁺).

N,N-diethyl-6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-amine (23):(MW-033): Product 23 was synthesized using the same ‘Suzuki’ reactionprotocol for 15 as before but using 8-4c. Compound 6-chloro-N,N-diethyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4c) (0.490 g, 1.8 mmol, 1eq) and 1-napthylboronic acid (0.440 g, 2.56 mmol, 1.37 eq) wassuspended in dimethoxyethane (DME) and water mixture (10:1 v/v) andfollowed the above protocol as described for 15. The product 23 waspurified by silica gel (200-400 mesh) column chromatography using ethylacetate and hexane (1:4 v/v) eluent. Product 23 obtained as pale yellowpowder (470 mg) in 69% (gravimetric) yield. ¹H NMR (500 MHz, CDCl₃): δ8.35 (dd, J=1.2, 4.8 Hz, 2H); 7.82 (dd, J=2.7, 8.0 Hz, 2H); 7.74 (d,J=8.4 Hz, 1H); 7.43-7.26 (m, 4H); 6.99 (dd, J=1.5, 4.5 Hz, 2H); 6.76 (s,1H); 3.76 (dd, J=7.0, 14.1 Hz, 4H); 1.34 (t, J=7.05, 7.05 Hz, 6H). HPLC(tr/purity): 15.01 min>96% (method A); exhibited a mass (ESI) m/z(MeOH): 355.20 (MH⁺).

6-chloro-N-methyl-N-propyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4d):Product 8-4d was made following the same protocol described above for8-4a, using compound 8-3 (0.600 g, 2.6 mmol) and N-methylpropane-1-amine(1.16 g, 15.9 mmol) 1-butanol and heated (110° C.) for 17 h. Product8-4d was obtained as a yellow powder (510 mg) in 79% (gravimetric)yield, with an apparent HPLC (tr/purity): 14.9 min>96% (HPLC method A),and a mass (ESI) m/z (MeOH)=263.10 (MH⁺).

N-methyl-6-(naphthalen-1-yl)-N-propyl-5-(pyridin-4-yl)pyridazin-3-amine(24): (MW-010): Product 24 was synthesized using the same ‘Suzuki’reaction protocol for 15 as before but using 8-4d. Compound6-chloro-N-methyl-N-propyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4d)(0.490 g, 1.8 mmol) and 1-napthylboronic acid (0.440 g, 2.56 mmol) wassuspended in dimethoxyethane (DME) and water mixture (10:1 v/v). Theproduct 24 was purified by column chromatography on silica gel (200-400mesh) using ethyl acetate: hexane (2:3 v/v) eluent. The final product 24was obtained as whitish powder (460 mg) in 69% yield (gravimetric). ¹HNMR (500 MHz, CDCl₃): δ 8.35 (dd, J=1.6, 4.57 Hz, 2H); 7.82 (d, J=8.1Hz, 2H); 7.71 (d, J=8.4 Hz, 1H); 7.43-7.26 (m, 4H); 6.99 (dd, J=1.6, 4.5Hz, 2H); 6.79 (s, 1H); 3.71 (t, J=7.4, 7.4 Hz, 2H); 3.29 (s, 3H); 1.79(m, 2H); 1.03 (t, J=7.35, 7.45 Hz, 3H). HPLC (tr/purity): 15.08min, >96% (method A); exhibited a mass (ESI) m/z (MeOH): 355.10 (MH⁺).

6-chloro-N-isopropyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4e): Product8-4e was made following the same protocol described above for 8-4a,using compound 8-3 (0.600 g, 2.6 mmol) and isopropyl-amine (0.939 g,15.9 mmol, 6 eq) in 1-butanol and heated (120° C.) for 25 h. The product8-4e was purified by column chromatography on silica gel (200-400 mesh)and eluted with ethyl acetate: hexane (9:1 v/v) followed bycrystallization from ethyl acetate and hexane to give the desiredproduct 8-4e as pale yellow crystals (445 mg) in 59% yield, with anapparent HPLC purity of 97% and exhibited a mass (ESI) m/z (MeOH)=249.00(MH⁺).

N-isopropyl-6-(naphthalen-1-yl)-5-(pyridin-4-yl)pyridazin-3-amine (25):(MW-031): Product 25 was synthesized using the same ‘Suzuki’ reactionprotocol for 15 as before. Compound6-chloro-N-isopropyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4e) (0.370 g,1.48 mmol, 1 eq) and 1-napthylboronic acid (0.350 g, 2.03 mmol, 1.37 eq)was suspended in dimethoxyethane (DME) and water mixture (10:1 v/v) andfollowed the same above procedure. The product 25 was purified by columnchromatography on silica gel (200-400 mesh) using ethyl acetate: hexane(2:3 v/v) eluent. The final product 25 was obtained as light yellowpowder (279 mg) in 54% yield (gravimetric). ¹H-NMR (500 MHz, CDCl₃): δ8.34 (dd, J=1.55, 6.05 Hz, 2H); 7.83 (d, J=8.2 Hz, 2H); 7.67 (d, J=8.4Hz, 1H); 7.43-7.26 (m, 4H); 6.95 (dd, J=1.6, 6.1, 2H); 6.70 (s, 1H);5.05 (b, 1H); 4.20 (m, 1H); 1.37 (d, J=6.4 Hz, 6H). HPLC (tr/purity):12.9 min, >97% (method A); exhibited a mass (ESI) m/z (MeOH): 341.10(MW).

N-isopropyl-6-(naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3-amine (26):MW-025): Product 26 was synthesized using the same ‘Suzuki’ reactionprotocol for 15 as before. Compound6-chloro-N-isopropyl-5-(pyridin-4-yl)pyridazin-3-amine (8-4e) (0.500 g,1.8 mmol, 1 eq) and 2-napthylboronic acid (0.426 g, 2.47 mmol, 1.37 eq)was suspended in dimethoxyethane (DME) and water mixture (10:1 v/v) andfollowed the same above procedure. The product 26 was purified by columnchromatography on silica gel (200-400 mesh) using ethyl acetate: hexane(3:2 v/v) eluent and product 51 obtained as white crystals (390 mg) in57% (gravimetric) yield. ¹H NMR (500 MHz, CDCl₃): δ 8.54 (dd, J=1.55,4.5 Hz, 2H); 7.92 (s, 1H); 7.80-7.68 (m, 3H); 7.49-7.31 (m, 3H); 7.13(dd, J=1.55, 4.0 Hz, 2H); 6.61 (s, 1H); 4.92 (b, 1H, —NH); 4.22 (m, 1H);1.36 (d, J=6.4 Hz, 6H). HPLC (tr/purity): 13.3 min>96% (method A) andexhibited a mass (ESI) m/z (MeOH): 341.10 (MH⁺).

3-chloro-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine (8-4f):Product 8-4f was made following the same protocol described above for8-4a, using compound 8-3 (0.750 g, 3.3 mmol) and 1-methylpiperazine (1.4g, 13.2 mmol) in 1-butanol and heated (120° C.) for 17 h. The product8-4f was purified by column chromatography on silica gel (200-400 mesh)and eluted with 2% methanol and ethyl acetate to give the desiredproduct 8-4f as a beige powder (620 mg) in 78% yield (gravimetric),purity By HPLC >93%; with a mass (ESI) of m/z (MeOH)=290.71 (MH⁺).

6-(4-methylpiperazine-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(27): (MW-150): Product 27 was synthesized using the same ‘Suzuki’reaction protocol for 15 as before but using 8-4f. Compound3-chloro-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine (8-4f)(0.187 g, 0.6 mmol, 1 eq) and 2-napthylboronic acid (0.156 g, 0.9 mmol,1.3 eq) was suspended in dimethoxyethane (DME) and water (10:1 v/v) in aheavy wall pressure vessel and purged with argon for 5 min. SubsequentlyTetrakis(triphenylphosphine) palladium (67 mg, 9 mol %), Na₂CO₃ (0.212g, 2.0 mmol) were added under argon, and followed the same protocolmentioned above. The product 27 was purified by column chromatography onsilica gel (200-400 mesh) using dichloromethane and 5% methanol followedby crystallization with ethyl acetate. The product 27 obtained as beigecolor crystals in 68% (gravimetric) yield; MP: 187.5-188° C.(uncorrected). ¹H NMR (500 MHz, CDCl₃): δ 8.55 (dd, J=1.55, 4.45 Hz,2H); 7.94 (s, 1H); 7.80-7.69 (m, 3H); 7.49-7.34 (m, 3H); 7.16 (dd,J=1.5, 4.5 Hz, 2H); 6.88 (s, 1H); 3.83 (t, J=4.8, 4.8, 4H); 2.62 (t,J=4.9, 4.9, 4H); 2.39 (s, 3H). HPLC (tr/purity): 11.26 min>97% (methoda); ESI m/z (MeOH): 382.2 (MH⁺); HRMS 381.1949 (calculated for C₂₄H₂₃N₅381.1953).

5-(6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazin-3-yl)-1H-indole(28): (MW-118): Product 28 was synthesized using the same ‘Suzuki’reaction protocol for 15 as before but using 8-4f. Compound3-chloro-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine (8-4f)(0.5 g, 1.7 mmol) and (1H-indol-5-yl)boronic acid (0.380 g, 2.3 mmol)was suspended in dimethoxyethane (DME) and water (10:1 v/v) and followedthe same protocol mentioned above. The product 28 was purified by columnchromatography on silica gel (200-400 mesh) with product elution usingethyl acetate and methanol (5%) followed by crystallization with ethylacetate. The product 28 obtained as fine crystals (465 mg) in 57%(gravimetric) yield; ¹H-NMR (500 MHz, DMSOd6): δ 11.16 (b, 1H); 8.49 (d,J=5 Hz, 2H); 7.49 (d, J=5 Hz, 1H); 7.34 (m, 1H); 7.28-7.23 (m, 4H); 6.93(dd, J=5.10 Hz, 1H); 6.37 (bs 1H); 3.70 (m, 4H); 2.67 (m, 4H); 2.25 (s,3H). HPLC (tr/purity): 4.40 min>97% (HPLC method A); exhibited a mass(ESI) of m/z (MeOH): 371.10 (MH⁺); HRMS calculated for C₂₂H₂₂N₆370.19059 found 370.1909.

4-(6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazin-3-yl)-1H-indole(29): (MW-108): Product 29 was synthesized using the same ‘Suzuki’reaction protocol for 15 as before but using 8-4f. Compound3-chloro-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine (8-4f)(130 mg, 0.44 mmol) and (1H-indol-4-yl)boronic acid (98 mg, 0.61 mmol)was suspended in dimethoxyethane (DME) and water (10:1 v/v) and followedthe same protocol mentioned above. The product 29 was purified by columnchromatography on silica gel (200-400 mesh) with product elution using5% methanol and ethyl acetate followed by crystallization with ethylacetate. The product 29 obtained as light yellow color powder (69 mg) in50% (gravimetric) yield; ¹H NMR (500 MHz, CD₃OD): δ 8.30 (d, J=5 Hz,2H); 7.42-7.36 (m, 2H); 7.25 (dd, J=5.5 Hz, 2H); 7.15 (d, J=5 Hz, 1H);7.09 (t, J=5.10 Hz, 1H); 6.93 (dd, J=5.5 Hz, 1H); 6.09 (d, J=5 Hz, 1H);4.97 (b, 1H, —NH); 3.84 (t, J=5, 5 Hz, 4H); 2.67 (t, J=5, 5 Hz, 4H);2.40 (s, 3H). HPLC (tr/purity): 12.2 min>97% (method A); exhibited amass (ESI) m/z (MeOH): 371.10 (MH⁺).

3-chloro-6-(piperazin-1-yl)-4-(pyridin-4-yl)pyridazine (8-4g)

Product 8-4g was made following the same protocol described above for8-4a, using compound 8-3 (0.200 g, 0.88 mmol) and piperazine (0.266 g,3.0 mmol) in 1-butanol and heated (120° C.) for 17 h. The product 8-4gwas purified by column chromatography on silica gel (200-400 mesh) usingethyl acetate and 5% methanol eluent to give product 8-4g as a beigepowder (209 mg) in 89% yield (gravimetric), HPLC (tr/purity): 4.6min>93%; exhibited a mass (ESI) of m/z (MeOH)=276.00 (MH⁺).

4-(6-(piperazin-1-yl)-4-(pyridin-4-yl)pyridazin-3-yl)-1H-indole (30):(MW-126)

Product 30 was synthesized using the same ‘Suzuki’ reaction protocol for15. Compound 3-chloro-6-(piperazin-1-yl)-4-(pyridin-4-yl)pyridazine(8-4g) (200 mg, 0.72 mmol) and (1H-indol-4-yl)boronic acid (159 mg, 0.99mmol) was suspended in dimethoxyethane (DME) and water (10:1 v/v) andfollowed the same protocol mentioned above. The product 30 was purifiedby column chromatography on silica gel (200-400 mesh) using ethylacetate and methanol (5%) eluent, followed by crystallization with ethylacetate. The product 30 obtained as light yellow color powder (110 mg)in 45% (gravimetric) yield; ¹H NMR (500 MHz, CD₃OD): δ 8.30 (d, J=5 Hz,2H); 7.42-7.36 (m, 2H); 7.25 (dd, J=5.5 Hz, 2H); 7.14 (m, 2H); 6.94 (d,J=5.10 Hz, 1H); 6.09 (d, J=5 Hz, 1H); 3.82 (m, 4H); 3.02 (m, 4H); HPLCmethod A (tr/purity): 12.0 min>97%; ESI m/z (MeOH): 357.10 (MH⁺).

Example 8-1

3-Chloro-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine (8-4f):Following the published protocol (Bioorg. Med. Chem Lett. 17, 414-418),a solution of 3,6-dichloro-4-pyridine-4-yl)pyridazine (8-3, 0.750 g, 3.3mmol, 1 eq) and 1-methylpiperazine (1.4 g, 13.2 mmol) in 25 ml ethanolwere placed in a heavy wall pressure vessel (Chemglass, Vineland, N.J.)and refluxed for 17 h. The product was purified by column chromatographyon silica gel (200-400 mesh) and eluted with 2% methanol and ethylacetate providing product 2 (620 mg, 78%) as a beige powder. ¹H NMIR(300 MHz, CDCl₃): δ 8.75 (dd, J=4.4, 1.7 Hz, 2H), 7.38 (dd, J=4.4, 1.7Hz, 2H), 6.82 (s, 1H), 3.71 (m, 4H), 2.55 (m, 4H), 2.36 (s, 3H); LC/MSESI positive R_(t)=1.47 min>93%, m/z=290.7 (M+H)⁺.

3-(6-Fluoronaphthalen-2-yl)-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine(49): (MW-203): A mixture of compound 2 (46 mg, 0.16 mmol),(6-fluoronaphthalen-2-yl)boronic acid J. Org. Chem. 70, 611-623) (25 mg,0.13 mmol), tetrakis (triphenylphosphine) palladium (15 mg, 13 μmol),sodium carbonate (43 mg, 0.40 mmol), water (65 μl) and DME (0.65 ml) waspurged with nitrogen, sealed and heated to 80° C. for 14 h. Cooled to20° C. and ethyl acetate (50 ml) was added. The mixture was washed withbrine (25 ml), dried (MgSO₄) and evaporated to leave a crystalline solid(26 mg, 41%). ¹HNMR (300 MHz, CDCl₃) δ 8.56 (d, J=5.8 Hz, 2H), 7.94 (s,1H), 7.71 (dd, J=9.1 Hz, J=5.6 Hz, 1H), 7.64 (d, J=8.5 Hz, 1H), 7.39 (m,2H), 7.21 (ddd, J=8.8 Hz, J=8.8 Hz, J=2.6 Hz, 1H), 7.15 (dd, J=4.4 Hz,J=1.8 Hz, 2H), 6.87 (s, 1H), 3.82 (m, 4H), 2.61 (m, 4H), 2.39 (s, 3H);LC/MS ESI positive R_(t)=2.82 min>96%, m/z=400.09 (M+H)⁻; HRMScalculated for C₂₄H₂₂FN₅ 399.1859, found 399.1865.

3-(7-Fluoronaphthalen-2-yl)-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine(50): (MW-017): A mixture of compound 2 (46 mg, 0.16 mmol),(7-fluoronaphthalen-2-yl)boronic acid (J. Med. Chem. 49, 2222-2231) (25mg, 0.13 mmol), tetrakis(triphenylphosphine) palladium (15 mg, 13 μmol),sodium carbonate (43 mg, 0.40 mmol), water (65 μl) and DME (0.65 ml) waspurged with nitrogen, sealed and heated to 80° C. for 14 h. Cooled to20° C., water was added (0.5 ml) and the mixture was purified bypreparative RPHPLC. The product fractions were combined, treated withsaturated sodium bicarbonate solution (20 ml) and extracted with ethylacetate (2×50 ml). The organic extracts were combined, washed with brine(25 ml), dried (MgSO₄) and evaporated to leave a crystalline solid (28mg, 54%). ¹H NMR (300 MHz, CDCl₃): δ 8.56 (d, J=6.1 Hz, 2H), 7.89 (s,1H), 7.78 (dd, J=8.8 Hz, J=5.6 Hz, 1H), 7.69 (d, J=8.5 Hz, 1H),7.37-7.7.22 (m, 3H), 7.15 (dd, J=4.4 Hz, J=1.2 Hz, 2H), 6.88 (s, 1H),3.83 (m, 4H), 2.62 (m, 4H), 2.40 (s, 3H); LC/MS ESI positive R_(t)=2.79min>96%, m/z=400.16 (M+H)⁻; HRMS calculated for C₂₄H₂₂F N₅ 399.1859,found 399.1865.

3-(8-Fluoronaphthalen-2-yl)-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine(52): (MW-032): A mixture of compound 2 (46 mg, 0.16 mmol),2-(8-fluoronaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (49mg, 0.18 mmol), tetrakis(triphenylphosphine)palladium (18 mg, 16 μmol),sodium carbonate (43 mg, 0.40 mmol), water (65 μl) and DME (0.65 ml) waspurged with nitrogen, sealed and heated to 80° C. for 14 h. Cooled to20° C., water was added (0.5 ml) and the mixture was purified by RPHPLC.The product fractions were combined, treated with saturated sodiumbicarbonate solution (20 ml) and extracted with ethyl acetate (2×50 ml).The organic extracts were combined, washed with brine (25 ml), dried(MgSO₄) and evaporated to leave a crystalline solid (29 mg, 45%). ¹H NMR(300 MHz, CDCl₃) δ 8.55 (d, J=5.8 Hz, 2H), 8.16 (s, 1H), 7.73 (dd,J=8.6, 1.5 Hz, 1H), 7.58 (d, J=8.2 Hz, 1H), 7.42 (dd, J=8.4, 1.8 Hz,1H), 7.43-7.35 (m, 1H), 7.14 (dd, J=4.7, 1.5 Hz, 2H), 7.10 (m, 1H), 6.88(s, 1H), 3.83 (m, 4H), 2.62 (m, 4H), 2.39 (s, 3H); LC/MS ESI positiveR_(t)=2.78 min>96%, m/z=400.16 (M+H)⁺; HRMS calculated for C₂₄H₂₂FN₅399.1859, found 399.1867.

3-(5-Fluoronaphthalen-2-yl)-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine(51): (MW-044): A mixture of compound 2 (49 mg, 0.17 mmol),2-(5-fluoronaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (45mg, 0.17 mmol), tetrakis(triphenylphosphine)palladium (19 mg, 17 μmol),sodium carbonate (45 mg, 0.43 mmol), water (65 μl) and DME (0.65 ml) waspurged with nitrogen, sealed and heated to 80° C. for 14 h. Cooled to20° C., water was added (0.5 ml) and the mixture was purified by RPHPLC.The product fractions were combined, treated with saturated sodiumbicarbonate solution (20 ml) and extracted with ethyl acetate (2×25 ml).The organic extracts were combined, washed with brine (25 ml), dried(MgSO₄) and evaporated to leave a crystalline solid (19 mg, 28%). ¹H NMR(300 MHz, CDCl₃) δ 8.56 (d, J=5.9 Hz, 2H), 7.96 (dd, J=3.5, 5.0 Hz, 1H),7.51 (d, J=8.5 Hz, 1H), 7.42 (dd, J=8.5, 1.2 Hz, 2H), 7.40-7.34 (m, 1H),7.18-7.10 (m, 3H), 6.88 (s, 1H), 3.84 (m, 4H), 2.62 (m, 4H), 2.40 (s,3H); LC/MS ESI positive R_(t)=2.85 min>97%, m/z=400.16 (M+H)⁺; HRMScalculated for C₂₄H₂₂FN₅ 399.1859, found 399.1866.

3-(5-fluoronaphthalen-1-yl)-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine(55): (MW-063): A mixture of compound 2 (42 mg, 0.14 mmol),(5-fluoronaphthalen-1-yl)boronic acid (32 mg, 0.17 mmol), tetrakis(triphenylphosphine)palladium (16 mg, 14 μmol) and sodium carbonate (44mg (0.42 mmol) in water (60 μl) and dimethoxyethane (0.60 ml) was purgedwith nitrogen, sealed tightly and heated to 80° C. for 18 h. The mixturewas cooled to 20° C., filtered over celite (methanol wash) and purifiedby RPHPLC (method C). The product fractions were combined, treated withsaturated aqueous sodium bicarbonate solution (25 ml) and extracted withethyl acetate (2×25 ml). The extracts were washed with brine (25 ml),dried (MgSO₄) and evaporated to leave a light yellow solid (22 mg, 39%).¹H-NMR (300 MHz, CDCl₃): δ 8.36 (d, J=5.8 Hz, 2H), 8.11 (d, J=8.2 Hz,1H), 7.48-7.41 (m, 2H), 7.75-7.67 (m, 2H), 7.35-7.24 (m, 2H), 7.10 (dd,J=10.3 Hz, J=6.6 Hz, 1H), 6.96 (s, 1H), 6.95 (dd, J=4.4 Hz, J=1.7 Hz,2H), 3.87-3.82 (m, 4H), 2.67-2.60 (m, 4H), 2.40 (s, 3H); LC/MS method A:ESI MS (M+H)⁺=400; R_(t)=2.67 minutes.

3-(3-fluoronaphthalen-1-yl)-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine(53): (MW-059): A mixture of compound 2 (42 mg, 0.14 mmol),(3-fluoronaphthalen-1-yl)boronic acid (25 mg, 0.13 mmol),tetrakis(triphenylphosphine)palladium (16 mg, 14 μmol), sodium carbonate(44 mg, 0.42 mmol), water (60 μl) and dimethoxyethane (0.6 ml) waspurged with a nitrogen atmosphere, sealed and heated to 80° C. for 15 h.Purified by RPHPLC (direct injection, method C). Product fractions werecombined, treated with aqueous saturated sodium bicarbonate solution (25ml) and extracted with ethyl acetate (2×25 ml). The combined extractswere washed with brine, dried (MgSO₄) and evaporated to leave a solid (9mg, 17%). ¹H-NMR (300 MHz, CDCl₃): δ 8.38 (dd, J=4.7 Hz, J=1.5 Hz, 2H),7.76 (d, J=8.2 Hz, 1H), 7.59 (d, J=8.5 Hz, 1H), 7.48-7.40 (m, 2H),7.33-7.20 (m, 1H), 7.13 (dd, J=9.0 Hz, J=2.6 Hz, 1H), 6.98 (dd, J=5.0Hz, J=1.7 Hz, 2H), 6.96 (s, 1H), 3.87 (m, 4H), 2.65 (m, 4H), 2.42 (s,3H); LC/MS method A: ESI pos. (M+H)⁺=400, R_(t)=2.63 minutes.

3-(6-fluoronaphthalen-1-yl)-6-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)pyridazine(54): (MW-197): A mixture of compound 2 (46 mg, 0.16 mmol),(6-fluoronaphthalen-1-yl)boronic acid (25 mg, 0.13 mmol),tetrakis(triphenylphosphine)palladium (15 mg, 13 μmol), sodium carbonate(43 mg, 0.40 mmol), water (65 μl) and DME (0.65 ml) was purged withnitrogen, sealed and heated to 80° C. for 14 h. Cooled to 20° C.,methanol (1 ml) and water (1 ml) were added and the product was purifiedby RPHPLC (method). The product fractions were combined, washed withbrine (25 ml), dried (MgSO₄) and evaporated to leave a crystalline solid(23 mg, 44%). ¹H-NMR (300 MHz, CDCl₃): δ 8.36 (d, J=5.8 Hz, 2H), 7.76(d, J=8.2 Hz, 1H), 7.71 (dd, J=9.4 Hz, J=5.6 Hz, 1H), 7.44 (dd, J=9.6Hz, J=2.6 Hz, 1H), 7.39 (dd, J=7.9 Hz, J=7.9 Hz, 1H), 7.21 (dd, J=7.0Hz, J=0.8 Hz, 1H), 7.13 (ddd, J=10.9 Hz, J=9.1, Hz, J=2.6 Hz, 1H), 6.96(s, 1H), 6.95 (dd, J=4.7, J=2.0, 2H), 3.86 (m, 4H), 2.64 (m, 4H), 2.41(s, 3H); LC/MS method A: ESI pos. (M+H)⁺=400, R_(t)=2.64 minutes.

Example 8-2

Compound 57(6-(4-methylpiperidin-1-yl)-3-(naphthalen-1-yl)-4-(pyridin-4-yl)pyridazine)(also described herein as SRM-137B) can be prepared in a similar manneras described in Example 8 by using 4-methylpiperidine as the amine andby using 1-naphthylboronic acid as the boronic acid.

Example 8-3

Compound 59(3-(naphthalen-1-yl)-4-(pyridin-4-yl)-6-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine)(also described herein as SRM-203A) can be prepared in a similar manneras described in Example 8 by using 2-(piperazin-1-yl)pyrimidine as theamine and by using 1-naphthylboronic acid as the boronic acid.

Example 8-4

Compound 56(6-(4-methylpiperidin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine)(also described herein as SRM-137A) can be prepared in a similar manneras described in Example 8 by using 4-methylpiperidine as the amine andby using 2-naphthylboronic acid as the boronic acid

Example 8-5

Compound 60(3-(naphthalen-2-yl)-4-(pyridin-4-yl)-6-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine)(also described herein as SRM-203B) can be prepared in a similar manneras described in Example 8 by using 2-(piperazin-1-yl)pyrimidine as theamine and by using 2-naphthylboronic acid as the boronic acid.

Example 9

Synthesis of precursor (9-7a): (X=H)

2-(pyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one (9-3a): A solution of4-picoline (9-2a, 0.11 g, 1.1 mmol, 0.11 mL) in THF (7 mL) undernitrogen atmosphere at −78° C. was treated with LDA [freshly prepared bytreating diisopropylamine (0.12 g, 1.2 mmol, 0.17 mL) in THF (3 mL) with2.5 M n-BuLi (0.50 mL, 1.25 mmol) in hexanes for 30 minutes undernitrogen atmosphere in an ice bath] for 10 minutes. The mixture stirred60 minutes and was treated with neatN,O-dimethyl-2-naphthalenehydroxamic acid (9-1, 0.20 g, 0.93 mmol)dropwise over ten minutes. The mixture stirred an additional two hoursthen saturated aqueous ammonium chloride (2 mL) was added to the mixtureand stirring continued an additional two hours while the temperature ofthe mixture rose to 20° C. The reaction mixture was diluted with ethylacetate (50 mL) and washed with water (3×50 mL), brine (25 mL), dried(Na₂SO₄) and evaporated. The product was chromatographed on silica geleluted with a gradient of ethyl acetate in hexanes (1:1 to 1:2 to 1:3)to leave the purified product 9-3a as a cream colored crystalline solid(153 mg, 67%). ¹H-NMR (300 MHz, CDCl₃): δ 8.58 (dd, J=4.4 Hz, J=1.8 Hz,2H), 8.53 (s, 1H), 8.05 (dd, J=8.5 Hz, J=1.8 Hz, 1H), 8.00-7.84 (m, 3H),7.66-7.55 (m, 2H), 7.25 (m, 2H), 4.43 (s, 2H); ESI MS (M+H)⁺=248; HPLCmethod A R_(t)=3.37 minutes.

Ethyl 3-(pyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate (9-4a): To asolution of 2-(pyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one (9-3a, 0.15g, 0.61 mmol) in 1,4-dioxane (6 mL) under nitrogen atmosphere at 20° C.was added sodium hydride (60% in mineral oil, 29 mg, 0.73 mmol) and themixture stirred for an hour. Ethyl bromoacetate (0.12 g, 0.73 mmol, 81μL) was added all at once and the mixture stirred for an hour. Saturatedaqueous ammonium chloride (2 mL) was added and stirring continued for anhour. The mixture was diluted with ethyl acetate (30 mL), washed withwater (2×30 mL), brine (20 mL), dried (Na₂SO₄) and evaporated to an oilyresidue that was purified by silica gel chromatography eluted with agradient of ethyl acetate in hexanes (1:1 to 2:1) to leave the product9-4a as a light yellow viscous oil (121 mg, 60%). ¹H-NMR (300 MHz,CDCl₃): δ 8.53 (dd, J=4.4 Hz, J=1.5 Hz, 2H), 8.49 (s, 1H), 7.99 (dd,J=8.5 Hz, J=1.7 Hz, 1H), 7.92 (d, J=7.9 Hz, 1H), 7.87-7.82 (m, 2H),7.62-7.50 (m, 2H), 7.29 (dd, J=4.4 Hz, J=1.5 Hz, 2H), 5.27 (m, 1H), 4.12(q, J=7.0 Hz, 2H), 3.42 (dd, J=17.0 Hz, J=9.4 Hz, 1H), 2.78 (dd, J=17.0Hz, J=5.6 Hz, 1H), 1.21 (t, J=7.0 Hz, 3H); ESI MS (M+H)⁺=334; HPLCmethod A R_(t)=3.89 minutes.

6-(naphthalen-2-yl)-5-(pyridin-4-yl)-2,3,4,5-tetrahydropyridazin-3-one(9-5a): A solution of ethyl3-(pyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate (9-4a, 0.12 g, 0.36mmol) in ethanol (3 mL) was treated with hydrazine hydrate (0.25 g, 5mmol, 0.25 mL) and refluxed for 20 hours. The mixture was cooled to 20°C. and purified by reversed phase HPLC (method B). The product fractionswere treated with saturated sodium bicarbonate solution (50 mL) andextracted with ethyl acetate (2×50 mL). The combined extracts werewashed with brine (50 mL), dried (Na₂SO₄) and evaporated to leave 48 mg(44%) of beige solid 9-5a. ¹H-NMR (300 MHz, CDCl₃): δ 8.76 (bs, 1H),8.57 (d, J=5.3 Hz, 2H), 7.98 (dd, J=8.5 Hz, J=1.8 Hz, 1H), 7.95 (s, 1H),7.85 (d, J=8.5 Hz, 1H), 7.86-7.76 (m, 2H), 7.55-7.46 (m, 2H), 7.21 (dd,J=4.4 Hz, J=1.2 Hz, 2H), 4.68 (d, J=6.5 Hz, 1H), 3.10 (dd, J=17.0 Hz,J=7.7 Hz, 1H), 2.90 (dd, J=17.0 Hz, J=1.8 Hz, 1H); ESI MS (M+H)⁺=302;HPLC method A R_(t)=3.11 minutes.

6-(Naphthalen-2-yl)-5-(pyridin-4-yl)-2,3-dihydropyridazin-3-one (9-6a):A solution of 6-(naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3(2H)-one(9-5a, 25 mg, 0.083 mmol) in DMSO (0.5 mL) and water (0.02 mL) wastreated with N-bromosuccinimide (75 mg, 0.42 mmol). After a slightexotherm the solution was stirred 72 hours then purified directly byreversed phase HPLC (method B). The combined product fractions weretreated with saturated sodium bicarbonate solution (50 mL) and extractedwith ethyl acetate (2×25 mL). The combined extracts were washed withbrine, dried (Na₂SO₄) and evaporated to leave product 9-6a as a beigesolid (17 mg, 69%). ¹H-NMR (300 MHz, CDCl₃): δ 11.13 (bs, 1H), 8.55 (bs,2H), 7.80 (dd, J=9.1 Hz, J=2.1 Hz, 1H), 7.76-7.68 (m, 3H), 7.55-7.45 (m,2H), 7.16 (dd, J=8.8 Hz, J=2.0 Hz, 1H), 7.09 (d, J=5.9 Hz, 2H), 7.05 (s,1H); ESI MS (M+H)⁺=300; HPLC method A R_(t)=2.88 minutes.

6-Bromo-4-(pyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (9-7a):6-(Naphthalen-2-yl)-5-(pyridin-4-yl)-2,3-dihydropyridazin-3-one (9-6a,0.15 g, 0.50 mmol) was added to a solution of phosphorousoxybromide(0.62 g, 2.0 mmol) in acetonitrile (3 mL) and heated to reflux whilestirring for ten hours. The reaction mixture was cooled in an ice bath,treated with ice (2 g) and stirred for an hour. The mixture wasneutralized with solid sodium carbonate to a pH=10 followed by dilutionwith ethyl acetate (20 mL) and water (20 mL). The organic layer wasseparated, washed with brine (50 mL), dried (Na₂SO₄) and evaporated. Theproduct was purified by chromatography on silica gel eluted with ethylacetate in hexanes (3:1). The purified product 9-7a was isolated as acolorless foamy solid (0.15 g, 83%). ¹H-NMR (300 MHz, CDCl₃): δ 8.59(dd, J=4.4 Hz, J=1.8 Hz, 1H), 8.04 (d, J=1.2 Hz, 1H), 7.86-7.73 (m, 3H),7.72 (s, 1H), 7.58-7.47 (m, 2H), 7.33 (dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.15(dd, J=4.4 Hz, J=1.7 Hz, 2H); ESI MS (M+H)⁺=362, 364; HPLC method AR_(t)=3.71 minutes.

6-(azetidin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine: (31):(MW-146)

A solution of 6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(9-7a) (35 mg, 0.097 mmol) in 200 proof ethanol (1 mL) was treated withazetidine (17 mg, 0.29 mmol, 20 μL). The mixture was sealed and heatedto 85° C. for 15 hours then cooled to room temperature. The mixture wasdiluted with ethyl acetate (25 mL), washed with saturated sodiumbicarbonate (25 mL), water (25 mL) and brine (25 mL), dried (MgSO₄) andevaporated. The product 31 was triturated with hexane to leave a beigecrystalline solid (32 mg, 98%). ¹H-NMR (300 MHz, CDCl₃) δ 8.53 (dd,J=4.4 Hz, J=1.5 Hz, 2H), 7.90 (d, J=1.0 Hz, 1H), 7.82-7.77 (m, 1H),7.73-7.69 (m, 1H), 7.69 (d, J=8.8 Hz, 1H), 7.51-7.41 (m, 2H), 7.35 (dd,J=8.5 Hz, J=1.8 Hz, 1H), 7.13 (dd, J=4.4 Hz, J=1.5 Hz, 2H), 6.50 (s,1H), 4.28 (dd, J=7.7 Hz, J=7.7 Hz, 4H), 2.55 (m, 2H); ESI MS (M+H)⁺=339;HPLC method A R_(t)=2.70 minutes.

3-(naphthalen-2-yl)-4-(pyridin-4-yl)-6-(pyrrolidin-1-yl)pyridazine:(32): (MW-148): A solution of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (50 mg,0.14 mmol) in 200 proof ethanol (2 mL) was treated with pyrrolidine (40mg, 0.56 mmol, 46 μL). The mixture was sealed and heated to 85° C. for15 hours then cooled to room temperature. The mixture was diluted withethyl acetate (25 mL), washed with saturated sodium bicarbonate (25 mL),water (25 mL) and brine (25 mL), dried (MgSO₄) and evaporated. Theresidue was purified by chromatography on silica gel eluted with agradient of 75% ethyl acetate in hexane to 100% ethyl acetate. Theproduct 32 fractions were evaporated to leave a beige crystalline solid(41 mg, 83%). ¹H-NMR (300 MHz, CDCl₃) δ 8.54 (dd, J=4.7 Hz, J=1.8 Hz,2H), 7.92 (s, 1H), 7.82-7.77 (m, 1H), 7.73-7.69 (m, 1H), 7.69 (d, J=8.5Hz, 1H), 7.50-7.41 (m, 2H), 7.38 (dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.16 (dd,J=4.4 Hz, J=1.5 Hz, 2H), 6.60 (s, 1H), 3.65 (m, 4H), 2.12 (m, 4H); ESIMS (M+H)⁺=353; HPLC method A R_(t)=2.78 minutes.

4-(6-(naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3-yl)morpholine: (33):(MW-152): A solution of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (50 mg,0.14 mmol) and morpholine (60 mg, 0.69 mmol) in 95% ethanol was refluxedfor 18 h. The reaction mixture was diluted with ethyl acetate (25 mL)and washed with saturated sodium bicarbonate solution (25 mL), water(2×25 mL), brine (25 mL), dried (Na₂SO₄) and evaporated to a tan solid33 (50 mg, 97%). ¹H-NMR (300 MHz, CDCl₃): δ 8.56 (bs, 2H), 7.96 (s, 1H),(7.82-7.69 (m, 2H), 7.72 (s, 1H), 7.52-7.43 (m, 2H), 7.35 (dd, J=8.5 Hz,J=1.8 Hz, 1H), 7.15 (d, J=5.3 Hz, 1H), 6.87 (s, 1H), 3.92 (m, 4H), 3.77(m, 4H). LC/MS (method A) R_(t)=3.02 mins., purity>95%, (M+H⁺)=369.

3-(Naphthalen-2-yl)-6-(piperazin-1-yl)-4-(pyridin-4-yl)pyridazine: (34):(MW-154): A solution of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (50 mg,0.14 mmol) and piperazine (59 mg, 0.69 mmol) in 95% ethanol (1 mL) wasrefluxed for 18 h. The reaction mixture was diluted with ethyl acetate(25 mL) and washed with saturated sodium bicarbonate solution (25 mL),water (2×25 mL), brine (25 mL), dried (Na₂SO₄) and evaporated to a beigesolid 34 (50 mg, 97%). ¹H-NMR (300 MHz, CDCl₃): δ 8.56 (dd, J=4.7 Hz,J=1.7 Hz, 2H), 7.94 (bs, 1H), 7.82-7.69 (m, 2H), 7.70 (s, 1H), 7.52-7.43(m, 2H), 7.35 (dd, J=8.0 Hz, J=1.8 Hz, 1H), 7.18-7.12 (m, 2H), 6.93 (d,J=16.7 Hz, 1H), 4.19 (m, 2H), 3.83 (m, 2H), 3.42 (m, 2H), 3.18 (bs, 1H),2.79 (m, 2H). LC/MS (method A) R_(t)=2.63 mins., purity>90%, (M+H⁺)=368.

6-(4-methylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine:(27): (MW-150): A solution of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (9.0 g, 25mmol) in ethanol (150 mL) in a 500 mL round bottom flask was treatedwith N-methylpiperidine (12.4 g, 125 mmol, 13.8 mL) and heated to refluxfor 18 h. The solvent was evaporated in vacuo. The residue was dissolvedin ethyl acetate (300 mL) and washed with water (2×200 mL) and saturatedsodium bicarbonate solution (100 mL). The product precipitated from theorganic layer and was collected via filtration on fritted glass. Theproduct was washed with water (20 mL) and ethyl acetate (50 mL) anddried under high vacuum to leave 8.6 g (90%) of 27 as light yellowsolid. ¹H-NMR (300 MHz, CDCl₃): δ 8.54 (dd, 2H, J=4.4 Hz, J=1.7 Hz),7.94 (s, 1H), 7.82-7.68 (m, 3H), 7.51-7.42 (m, 2H), 7.35 (dd, 1H, J=8.5Hz, J=1.8 Hz), 7.15 (dd, 2H, J=4.4 Hz, J=1.7), 6.88 (s, 1H), 3.82 (dd,4H, J=5.2 Hz, J=5.0 Hz), 2.60 (dd, 4H, J=5.2 Hz, J=5.0 Hz), 2.39 (s,3H); ESI MS (M+H)⁺=382; HPLC method A R_(t)=2.58 minutes.

N-cyclopropyl-6-(naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3-amine:(35): (MW-153): A solution of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (50 mg,0.14 mmol) and cyclopropylamine (39 mg, 0.69 mmol) in 95% ethanol wasrefluxed for 18 h in a closed vial. LC/MS analysis (method A) indicatedthe starting material was half converted to product. Additionalcyclopropylamine (39 mg, 0.69 mmol) was added and the mixture wasrefluxed 18 h. The reaction mixture was diluted with ethyl acetate (25mL) and washed with saturated sodium bicarbonate solution (25 mL), water(2×25 mL), brine (25 mL), dried (Na₂SO₄) and evaporated. The product 35was purified by RPHPLC (method B) and the product fractions werecombined, diluted with saturated aqueous sodium bicarbonate solution andextracted with ethyl acetate (2×25 mL). The combined extracts werewashed with brine (25 mL), dried (Na₂SO₄) and evaporated to a lightbrown solid 35 (37 mg, 76%). ¹H-NMR (300 MHz, CDCl₃): δ 8.56 (bs, 2H),7.93 (s, 1H), 7.82-7.72 (m, 2H), 7.69 (s, 1H), 7.52-7.43 (m, 2H), 7.32(dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.17 (d, J=5.9 Hz, 2H), 7.04 (s, 1H), 6.01(bs, 1H), 2.72-2.62 (m, 1H), 0.97-0.90 (m, 2H), 0.77-0.68 (m, 2H). LC/MS(method A) R_(t)=2.88 mins., purity>95%, (M+H⁺)=339.

6-(1-methylpiperidin-4-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine:(36): (MW-164): Was made in two steps as follows

Step-I:6-(1-Methyl-1,2,3,6-tetrahydropyridin-4-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine:A solution of 6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(9-7a) (100 mg, 0.28 mmol),1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,6-tetrahydropyridinemonohydrochloride (110 mg, 0.42 mmol), Pd(dppf) dichloromethane adduct(22 mg, 28 μmol) and potassium carbonate (116 mg, 0.84 mmol) was purgedwith nitrogen gas then diluted with dioxane (3 mL) and water (0.4 mL).The mixture was purged with nitrogen gas again, tightly capped andheated to 80° C. for 18 h. The mixture was cooled to 20° C., dilutedwith ethyl acetate (50 mL), washed with water (50 mL), brine (50 mL),dried (Na₂SO₄) and evaporated. The crude product was purified by RPHPLC(method B) and the product fractions were combined and treated withsaturated aqueous sodium bicarbonate solution. The product was extractedwith ethyl acetate (2×25 mL) and the combined extracts were washed withbrine (25 mL), dried (Na₂SO₄) and evaporated to leave a light tan solid(100 mg, 94%). ¹H-NMR (300 MHz, CDCl₃): δ 8.58 (dd, J=4.4 Hz, J=1.7 Hz,2H), 8.08 (bs, 1H), 7.82-7.73 (m, 2H), 7.75 (s, 1H), 7.60 (s, 1H),7.55-7.45 (m, 2H), 7.41 (dd, J=8.5 Hz, J=1.8 Hz, 1H), 7.19 (dd, J=4.4Hz, J=1.8 Hz, 2H), 6.82 (m, 1H), 3.29 (m, 2H), 2.98 (m, 2H), 2.80 (dd,J=5.6 Hz, J=5.8 Hz, 2H), 2.49 (s, 3H). LC/MS (method A) R_(t)=2.73mins., purity>95%, (M+H⁺)=379.

Step-II:6-(1-methylpiperidin-4-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine:6-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (100 mg,0.26 mmol) was hydrogenated (45 psi) in methanol (5 mL) over 10% Pd/C(20 mg) for 48 h with agitation. The catalyst was filtered and thesolvents evaporated. The crude product was purified by RPHPLC to leave14 mg (14%) of beige solid 36. ¹H-NMR (300 MHz, CDCl₃): δ 8.30 (d, J=5.9Hz, 2H), 8.06 (bs, 1H), 7.82-7.73 (m, 2H), 7.76 (s, 1H), 7.57-7.48 (m,2H), 7.43 (s, 1H), 7.38 (dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.16 (dd, J=4.4Hz, J=1.8 Hz, 2H), 3.23 (m, 4H), 2.52 (s, 3H), 2.44 (m, 1H), 2.22 (m,4H). LC/MS (method A) R_(t)=2.69 mins., purity>90%, (M+H⁺)=381.

6-Cyclopropyl-4-(pyridin-4-yl)-3-(naphthalen-2-yl)pyridazine: (9):(MW-125): A mixture of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (40 mg,0.11 mmol), cyclopropyl boronic acid (13 mg, 0.15 mmol),PdCl₂(dppf)·CH₂Cl₂ (8 mg, 0.011 mmol), silver oxide (64 mg, 0.28 mmol),potassium carbonate (46 mg, 0.33 mmol) and 1,4-dioxane (1 mL) wasstirred and purged with nitrogen. The mixture was heated to 80° C. for18 hours. The reaction mixture was cooled to 20° C. and filtered throughcelite (ethyl acetate wash). The filtrate was evaporated to dryness andthe crude product was purified by RPHPLC (method B). The productfraction was diluted with saturated sodium bicarbonate solution (50 mL)and the product was extracted with ethyl acetate (2×25 mL). The combinedorganic layers were washed with brine (2×25 mL), dried (Na₂SO₄) andevaporated to leave the product 9 as a white solid (16 mg, 45%). ¹H-NMR(300 MHz, CD₃OD): δ 8.46 (d, J=4.4 Hz, 2H), 7.93 (d, J=1.5 Hz, 1H),7.88-7.77 (m, 4H), 7.65 (s, 1H), 7.56-7.46 (m, 2H), 7.37 (dd, J=8.5 Hz,J=1.7 Hz, 1H), 7.33 (dd, J=4.7 Hz, J=1.5 Hz, 1H), 2.4-2.35 (m, 1H),1.30-1.22 (m, 4H); ESI MS (M+H)⁺=324; HPLC method A R_(t)=3.47 minutes.

Synthesis of precursor (9-7b): (X=F)

2-(3-fluoropyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one (9-3b): Asolution of 3-fluoro-4-picoline (9-2b, 3.2 g, 29 mmol, 2.9 mL) in THF(200 mL) under nitrogen atmosphere at −78° C. was treated with LDA[freshly prepared by treating diisopropylamine (5.3 g, 53 mmol, 7.4 mL)in THF (50 mL) with 2.5 M n-BuLi in hexanes for 30 minutes undernitrogen atmosphere in an ice bath] for 10 minutes. The mixture stirred60 minutes and was treated with neatN,O-dimethyl-2-naphthalenehydroxamic acid (9-1, 7.5 g, 35 mmol) dropwiseover ten minutes. The mixture stirred an additional two hours thensaturated aqueous ammonium chloride (20 mL) was added to the mixture andstirring continued an additional two hours while the temperature of themixture rose to 20° C. Approximately 200 mL of solvent was removed fromthe mixture in vacuo and the residue was diluted with ethyl acetate (200mL) and washed with water (3×100 mL). The product was extracted with 1NHCl (6×75 mL) and the combined acidic extracts were neutralized withsolid sodium carbonate to pH=9. The product was extracted with ethylacetate (2×100 mL) and the extracts were dried (Na₂SO₄) and evaporated.The product 9-3 was obtained as a light yellow crystalline solid (4.4 g,57%). ¹H-NMR (300 MHz, CDCl₃): δ 8.57 (s, 1H), 8.50 (d, J=1.5 Hz, 1H),8.41 (dd, J=4.6 Hz, J=1.0 Hz, 1H), 8.07 (dd, J=8.2 Hz, J=1.5 Hz), 8.01(d, J=18.7 Hz, 1H), 7.94 (m, 2H), 7.68-7.57 (m, 3H), 4.52 (s, 2H); ESIMS (M+H)⁺=266; HPLC method A R_(t)=4.29 minutes.

Ethyl 3-(3-fluoropyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate(9-4b): To a solution of2-(3-fluoropyridin-4-yl)-1-(naphthalen-2-yl)ethan-1-one (9-3b, 4.4 g, 17mmol) in 1,4-dioxane (150 mL) under nitrogen atmosphere at 20° C. wasadded sodium hydride (60% in mineral oil, 0.80 g, 20 mmol) and themixture stirred for an hour. Ethyl bromoacetate (3.3 g, 20 mmol, 2.2 mL)was added all at once and the mixture stirred for 18 hours. Saturatedaqueous ammonium chloride (20 mL) was added and stirring continued foran hour. The mixture was evaporated in vacuo to approximately 50 mL involume and diluted with ethyl acetate (200 mL). The mixture was washedwith water (2×100 mL), brine (50 mL), dried (Na₂SO₄) and evaporated toan oily residue that was purified by silica gel chromatography elutedwith a gradient of hexanes/ethyl acetate (3:1 to 2:1) to leave theproduct 9-4b as a light yellow viscous oil (4.4 g, 74%). ¹H-NMR (300MHz, CDCl₃): δ 8.54 (d, J=1.4 Hz, 1H), 8.46 (d, J=1.4 Hz, 1H), 8.31 (dd,J=5.0 Hz, J=0.8 Hz, 1H), 8.00 (dd, J=8.5 Hz, J=1.6 Hz, 1H), 9.94 (bd,J=8.2 Hz, 1H), 7.88-7.82 (m, 2H), 7.62-7.51 (m, 2H), 7.22 (dd, J=5.0 Hz,J=5.0 Hz, 1H), 5.62 (dd, J=9.4 Hz, J=5.3 Hz, 1H), 4.13 (q, J=7.0 Hz,2H), 3.40 (dd, J=17.1 Hz, J=9.4 Hz, 1H), 2.80 (dd, J=17.1 Hz, J=5.3 Hz,1H), 1.21 (t, J=7.0 Hz); ESI MS (M+H)⁺=352; HPLC method A R_(t)=5.38minutes.

6-(naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3,4,5-tetrahydropyridazin-3-one(9-5b): A solution of ethyl3-(3-fluoropyridin-4-yl)-4-(naphthalen-2-yl)-4-oxobutanoate (9-4b, 4.4g, 12.5 mmol) in ethanol (32 mL) and acetic acid (1.5 mL) was treatedwith hydrazine hydrate (12.5 g, 250 mmol, 12.5 mL) and refluxed for 20hours. The mixture was cooled to 20° C., water (5 mL) was added and thebeige precipitate was filtered, washed with 25% aqueous ethanol andair-dried to leave the product 9-5b as 1.85 g (46%). ¹H-NMR (300 MHz,CDCl₃): δ 8.92 (bs, 1H), 8.55 (d, J=1.5 Hz, 1H), 8.30 (d, J=5.0 Hz, 1H),7.99 (dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.90 (d, J=1.5 Hz, 1H), 7.88-7.78 (m,3H), 7.55-7.45 (m, 2H), 7.04 (dd, J=6.5 Hz, J=5.0 Hz, 1H), 5.02 (dd,J=7.9 Hz, J=1.5 Hz, 1H), 3.10 (dd, J=17.3 Hz, J=7.9 Hz, 1H), 2.90 (dd,J=17.3 Hz, J=1.5 Hz, 1H); ESI MS (M+H)⁺=320; HPLC method A R_(t)=4.21minutes.

6-(Naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3-dihydropyridazin-3-one(9-6b): A solution of6-(naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3,4,5-tetrahydropyridazin-3-one(9-5b, 3.2 g, 10 mmol) in DMSO (80 mL) and water (1.6 mL) was treatedwith N-bromosuccinimide (8.9 g, 50 mmol). After a slight exotherm thesolution was stirred 72 hours then diluted with water (300 mL) andextracted with ethyl acetate (3×150 mL). The combined organic layerswere washed with water (200 mL), saturated sodium bicarbonate solution(150 mL) and brine (50 mL). The solution was dried (Na₂SO₄) andevaporated. The solid residue was stirred rapidly in saturated sodiumbicarbonate solution (50 mL) and water (100 mL) for 18 hours thenfiltered, washed with water (5×25 mL) and dried under vacuum to leavethe product as a tan powder (3.1 g, 97%). ¹H-NMR (300 MHz, CDCl₃): δ11.69 (bs, 1H), 8.42 (dd, J=5.0 Hz, J=5.0 Hz, 1H), 8.38 (d, J=0.9 Hz,1H), 7.80 (d, J=7.7 Hz, 1H), 7.74-7.67 (m, 3H), 7.55-7.44 (m, 2H), 7.26(dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.20-7.13 (m, 1H), 7.11 (s, 1H); ESI MS(M+H)⁺=318; HPLC method A R_(t)=4.07 minutes.

6-Bromo-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (9-7b):6-(Naphthalen-2-yl)-5-(3-fluoropyridin-4-yl)-2,3-dihydropyridazin-3-one(9-6b, 3.0 g, 9.5 mmol) was added to a solution of phosphorousoxybromide(11 g, 38 mmol) in acetonitrile (100 mL) and heated to reflux whilestirring for ten hours. The reaction mixture was cooled in an ice bath,treated with ice (50 g) and stirred for an hour. Approximately half ofthe solvent was removed in vacuo. The product mixture was diluted withethyl acetate (100 mL) and saturated sodium bicarbonate solution (300mL) was cautiously added with stirring until it reached a pH=8. Theorganic layer was separated, washed with brine (50 mL), dried (Na₂SO₄)and evaporated. The product was purified by chromatography on silica geleluted with a gradient of 40% to 50% ethyl acetate in hexanes. Thepurified product 9-7b was isolated as a colorless foamy solid (2.2 g,61%). ¹H-NMR (300 MHz, CDCl₃): δ 8.47 (d, J=1.2 Hz, 1H), 8.41 (dd, J=5.0Hz, J=1.0 Hz, 1H), 7.96 (d, J=1.5 Hz, 1H), 7.85-7.73 (m, 4H), 7.57-7.46(m, 2H), 7.41 (dd, J=8.8 Hz, J=2.1 Hz), 7.15 (dd, J=5.3 Hz, J=4.7 Hz,1H); ESI MS (M+H)⁺=380, 382; HPLC method A R_(t)=5.29 minutes.

6-Cyclopropyl-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazine(10): (MW-167): A mixture of6-Bromo-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazine (9-7b,2.2 g, 5.8 mmol), cyclopropyl boronic acid (0.70 g, 8.1 mmol),PdCl₂(dppf)·CH₂Cl₂ (0.42 g, 0.58 mmol), silver oxide (3.36 g, 14.5mmol), potassium carbonate (2.4 g, 17.4 mmol) and 1,4-dioxane (50 mL)was stirred and purged with nitrogen. The mixture was heated to 80° C.for 18 hours. The reaction mixture was cooled to 20° C. and filteredthrough celite (ethyl acetate wash). The filtrate was evaporated todryness and the crude product was purified by chromatography on silicagel eluted with 50% ethyl acetate in hexanes to leave the product 10 asa gum that crystallized upon trituration with hexane (1.1 g, 56%).¹H-NMR (300 MHz, CDCl₃): δ 8.45 (d, J=1.4 Hz, 1H), 8.38 (dd, J=4.9 Hz,J=1.0 Hz, 1H), 7.95 (m, 1H), 7.84-7.51 (m, 2H), 7.74 (s, 1H), 7.54-7.43(m, 3H), 7.33 (d, J=1.0 Hz, 1H), 7.14 (dd, J=5.6 Hz, J=5.0 Hz, 1H), 2.27(m, 1H), 1.38 (m, 2H), 1.24 (m, 2H); ESI MS (M+H)⁺=342; HPLC method AR_(t)=4.94 minutes.

4-(3-fluoropyridin-4-yl)-6-(4-methylpiperazin-1-yl)-3-(naphthalen-2-yl)pyridazine:(37): (MW-149): A solution of6-bromo-4-(3-fluoropyridin-4-yl)-3-(naphthalen-2-yl)pyridazine 9-7b (50mg, 0.13 mmol) and N-methylpiperazine (66 mg, 0.66 mmol) in 95% ethanol(1 mL) was heated to reflux for 18 h. The mixture was diluted with ethylacetate (25 mL), washed with saturated sodium bicarbonate solution (25mL), water (2×25 mL), brine (25 mL), dried (Na₂SO₄) and evaporated to alight tan solid 37 (50 mg, 96%). ¹H-NMR (300 MHz, CDCl₃): δ 8.44 (d,J=1.5 Hz, 1H), 8.36 (d, J=5.0 Hz, 1H), 7.87 (s, 1H), 7.82-7.69 (m, 2H),7.72 (s, 1H), 7.51-7.42 (m, 3H), 7.13 (dd, J=6.1 Hz, J=4.7 Hz, 1H), 6.95(s, 1H), 3.95 (bm, 4H), 2.77 (bm, 4H), 2.49 (s, 3H). LC/MS (method A)R_(t)=3.40 mins., purity>95%, (M+H⁺)=400.

Formation of Salt forms of the base to Hydrochloride hydrates:

Hydrochloride hydrate of products of the base: 7, 9, 15, 16, and 25-27:This was done as described in Hu et al, Bioorg Med Chem Lett 2007, 17:414-418. Approximately 1 mmol of the respective compound was suspendedin 8.2 mL of anhydrous isopropanol (99.5%, Aldrich), heated to 85° C.with stirring until dissolved, then 3.1 equiv (234 μL) of concentratedultrapure HCl (12N, JT Baker Ultrex® II, Product 6900-05) was added tothe clear solution, resulting in immediate generation of a suspension ofsolids. The suspension was stirred for 10 min (80° C.), allowed to coolto ambient temperature, then the vessel placed on ice for ˜2.5 h, thenstored (˜16 hr) at 4° C. The resulting yellow precipitate was filteredon a medium frit sintered glass funnel under vacuum, washed (3×) withcold anhydrous isopropanol followed by three washes with cold anhydrousether, and dried under vacuum. The precipitate was stored in vacuo in aglass desiccator containing silica gel until a constant weight wasattained. The final products were obtained in approximately 82% yield(gravimetric) compared to the starting material. Hydrochloride hydrateformation was confirmed by elemental analysis. The goal was to obtain amole ratio of HCl:compound that is >1. Under these conditions for thestudies described here, elemental analysis indicated a ratio of ˜2.

(MW-077 hydrochloride hydrate: (8): EA calculated for C₂₂H₂₃C₂N₃O₂: C,61.12; H, 5.36; N, 9.72; Cl, 16.40; O, 7.40; experimentally found C,59.54; H, 5.38; N, 9.38; Cl, 15.49; O, 9.46.

MW-156 hydrochloride hydrate: (38): EA calculated for C₂₀H₂₂C₂N₄O₂: C,57.01; H, 5.26; Cl, 16.83; N, 13.30; O, 7.59; experimentally found C,58.83; H, 5.56; Cl, 11.87; N, 13.11; O, 5.42.

MW-200 hydrochloride hydrate: (39): EA calculated for C₂₀H₂₂Cl₂N₄O₂: C,57.01; H, 5.26; Cl, 16.83; N, 13.30; O, 7.59; experimentally found C,55.48; H, 5.34; Cl, 16.34; N, 12.69; O, 4.06.

MW-031 hydrochloride hydrate: (40): EA calculated for C₂₂H₂₄Cl₂N₄O: C,61.26; H, 5.61; Cl, 16.44; N, 12.99; O, 3.71; experimentally found C,60.19; H, 5.65; Cl, 15.27; N, 12.83; O, 3.99.

MW-025 hydrochloride hydrate: (41): EA calculated for C₂₂H₂₆Cl₂N₄O₂: C,58.80; H, 5.83; Cl, 15.78; N, 12.47; O, 7.12; experimentally found C,58.33; H, 5.92; Cl, 15.19; N, 12.31; O, 6.43.

MW-150 hydrochloride hydrate: (42): EA calculated for C₂₄H₂₉Cl₂N₅O₂: C,58.78; H, 5.96; N, 14.28; Cl, 14.46; O, 6.52; experimentally found C,57.88; H, 5.72; N, 14.03; Cl, 14.31; O, 1.84.

MW-125 hydrochloride hydrate: (43): EA calculated for C₂₂H₂₃Cl₂N₃O₂: C,61.12; H, 5.36; N, 9.72; Cl, 16.40; O, 7.40; experimentally found C,61.15 H, 4.88; N, 9.58; Cl, 15.99; O, 8.01.

Example 9-1

3-(naphthalen-2-yl)-6-(piperidin-1-yl)-4-(pyridin-4-yl)pyridazine (61)(also referred to herein as MW-086) was prepared from compound 9-7a, thesynthesis of which is described above in Example 9.

3-(naphthalen-2-yl)-6-(piperidin-1-yl)-4-(pyridin-4-yl)pyridazine (61):(MW-086): A solution of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (25 mg, 69μmol) and piperidine (29 mg, 0.35 mmol, 34 μl) in ethanol (95%, 1 ml)was heated to reflux for 18 h. The mixture was purified by purified bypreparative RPHPLC. The product fractions were combined and neutralizedwith saturated aqueous sodium bicarbonate solution (25 ml) and theproduct was extracted with ethyl acetate (2×25 ml). The extracts werewashed with brine, dried (MgSO₄) and evaporated to leave 24 mg solid(95%). ¹H NMR (300 MHz, CDCl₃) δ 8.54 (d, J=5.2 Hz, 2H), 7.94 (s, 1H),7.80 (dd, J=5.6, 2.6 Hz, 1H), 7.75-7.67 (m, 2H), 7.50-7.40 (m, 2H), 7.37(dd, J=6.5, 1.8 Hz, 1H), 7.16 (dd, J=4.4, 1.4 Hz, 2H), 3.79 (bs, 4H),1.75 (bs, 6H); LC/MS R_(t)=1.85 min., purity>95%, m/z=367 (M+H)⁺.

Example 9-2

6-(4-ethylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(62) (also referred to as herein MW-026) was prepared from compound9-7a, the synthesis of which is described above in Example 9.

6-(4-ethylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(62) (MW-026): A solution of6-bromo-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (9-7a) (40 mg,0.11 mmol) in 95% ethanol (1 ml) was treated with N-ethylpiperazine (63mg, 0.55 mmol, 70 μl) and heated to reflux for 18 h. After cooling to20° C. ethyl acetate (25 ml) was added and the mixture was washed withwater (3×25 ml), dried (MgSO₄) and evaporated to a solid (43 mg, 99%).¹H-NMR (300 MHz, CDCl₃): δ 8.54 (d, J=5.6 Hz, 2H), 7.94 (s, 1H), 7.80(d, J=7.8 Hz, 1H), 7.78-7.68 (m, 2H), 7.52-7.41 (m, 2H), 7.36 (dd, J=8.5Hz, J=1.4 Hz, 1H), 7.15 (d, J=5.6 Hz, 2H), 6.87 (s, 1H), 3.83 (m, 4H),2.64 (m, 4H), 2.51 (q, J=7.0 Hz, 2H), 1.16 (t, J=7.0 Hz, 3H); LC/MSmethod A: ESI pos. (M+H)⁺=396, R_(t)=2.73 minutes.

Munoz L, Ralay Ranaivo H, Roy S M, Hu W, Craft J M, McNamara L K, WingChico L, Van Eldik L, and Watterson D M; A novel p38α MAPK inhibitorsuppresses brain proinflammatory cytokine up-regulation and attenuatessynaptic dysfunction and behavioral deficits in an Alzheimer's diseasemouse model. J Neuroinflammation 2007, 4: 21.

Schmitt M, de Araújo-Júnior J X, Oumouch S, Bourguignon J J; Use of4-bromo pyridazine 3,6-dione for building 3-amino pyridazine libraries.Molecular Diversity, 2006, 10: 429-434

Tamayo N, Liao L, Goldberg M, Powers D, Tudor Y, Yu V, Wong Lu Henkle B,Middleton, S, Syed R; Design and synthesis of potent pyridazineinhibitors of p38 MAP kinase. Bioorg Med Chem Lett 2005, 15: 2409-2413.

Example 10

N-methoxy-N-methyl-2-naphthamide (10-2): A mixture of 2-naphthoylchloride (20 g, 0.12 mol) and N,O-dimethylhydroxylamine hydrochloride(14 g, 0.14 mol) in dichloromethane (1 L) was stirred, cooled in an icebath and treated with diisopropylethylamine (39 g, 0.30 mol, 54 mL)dropwise over 30 minutes. The mixture warmed to 20° C. over 2 hours thenstirred an additional 16 hours. The solvents were evaporated and theresidual solid was dissolved in ethyl acetate (500 mL) and water (500mL). The organic layer was separated and washed with 1N HCl (300 mL),water (300 mL) and brine (200 mL). The organic phase was dried (Na₂SO₄)and evaporated to leave the product as a light tan oil (22.1 g, 86%).Mass ESI m/z 216 (M+1).

1-(Naphthalen-2-yl)-2-(pyridin-4-yl)ethan-1-one (10-3): A solution of4-picoline (7.8 g, 84 mmol, 8.1 mL) in anhydrous THF (500 mL) undernitrogen atmosphere at −78° C. was treated with a solution of freshlyprepared LDA [from a solution of diisopropyl amine (12.7 g, 126 mmol,17.7 mL) in THF (70 mL) under nitrogen atmosphere in an ice bath treatedwith n-butyllithium solution (2.5M in hexanes, 50 mL, 126 mmol) andstirred for 30 mins.] over 20 mins. The mixture stirred at −78° C. for 1hour and was treated with a solution of N-methoxy-N-methyl-2-naphthamide(10-2, 20 g, 93 mmol) in THF (80 mL) drop-wise over 1.5 hours whileensuring the temperature was maintained at or below −75° C. The mixturewas then allowed to warm to 20° C. over 4 hours and stirred anadditional 16 hours at this temperature. Saturated ammonium chloridesolution (50 mL) was added to the mixture and allowed to stir 30 mins.The solvents were evaporated in vacuo to approximately 20% of theoriginal reaction mixture volume and the residue was dissolved in ethylacetate (500 mL) and water (300 mL). The organic layer was separated andwashed with water (300 mL). The product was extracted from the organiclayer with 1N HCl (2×200 mL). The combined acid extracts wereneutralized with solid sodium bicarbonate to pH=8. The precipitatedproduct was filtered, washed with water (2×30 mL) and dried under vacuumfor 16 hours to leave a light yellow solid. ¹H-NMR (300 MHz, CDCl₃): δ8.58 (dd, J=4.4 Hz, J=1.8 Hz, 2H), 8.53 (s, 1H), 8.05 (dd, J=8.5 Hz,J=1.8 Hz, 1H), 8.00-7.84 (m, 3H), 7.66-7.55 (m, 2H), 7.25 (m, 2H), 4.43(s, 2H); ESI MS (M+H)⁺=248; HPLC method A R_(t)=3.37 minutes.

Ethyl 4-(naphthalen-2-yl)-4-oxo-3-(pyridin-4-yl)butanoate (10-4): Asolution of 1-(Naphthalen-2-yl)-2-(pyridin-4-yl)ethan-1-one (10-3, 17 g,69 mmol) in anhydrous 1,4-dioxane under nitrogen atmosphere was treatedwith sodium hydride (60% in mineral oil, 3.2 g, 79 mmol) and stirred for1 hour. The mixture, which now has a precipitate, was treated with ethyl2-bromoacetate (13 g, 79 mmol, 8.8 mL) and stirred for 18 hours. Asolution of saturated ammonium chloride (50 mL) was added and themixture stirred for 30 mins. The solvents were evaporated toapproximately 80% of the original volume and the residue was dilutedwith ethyl acetate (300 mL) and water (200 mL). The mixture was filteredthrough celite (ethyl acetate wash) to remove a small amount of yellowprecipitate. The organic layer was separated and the product wasextracted with 1N HCl (4×100 mL). The combined acid extracts wereneutralized with solid sodium carbonate (pH=9) and the product wasextracted with ethyl acetate (2×100 mL). The combined organic extractswere washed with brine, dried over Na₂SO₄ and evaporated. The residuewas chromatographed on silica gel (80 g column) eluted with a gradientof ethyl acetate-hexanes (1:1 to 2:1) to leave the product as a lightyellow solid (11.5 g, 50%). ¹H-NMR (300 MHz, CDCl₃): δ 8.53 (dd, J=4.4Hz, J=1.5 Hz, 2H), 8.49 (s, 1H), 7.99 (dd, J=8.5 Hz, J=1.7 Hz, 1H), 7.92(d, J=7.9 Hz, 1H), 7.87-7.82 (m, 2H), 7.62-7.50 (m, 2H), 7.29 (dd, J=4.4Hz, J=1.5 Hz, 2H), 5.27 (m, 1H), 4.12 (q, J=7.0 Hz, 2H), 3.42 (dd,J=17.0 Hz, J=9.4 Hz, 1H), 2.78 (dd, J=17.0 Hz, J=5.6 Hz, 1H), 1.21 (t,J=7.0 Hz, 3H); ESI MS (M+H)⁺=334; HPLC method A R_(t)=3.89 minutes.

6-(Naphthalen-2-yl)-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one(10-5): A solution ethyl4-(naphthalen-2-yl)-4-oxo-3-(pyridin-4-yl)butanoate (10-4, 16 g, 48mmol) in ethanol (125 mL) and acetic acid (5 mL) was treated withhydrazine hydrate (80 g, 0.96 mol, 80 mL) and refluxed for 20 hours. Aprecipitate formed. The mixture was cooled and concentrated underreduced pressure to 50% of the original volume. The mixture was cooledin an ice bath and filtered to collect the product which was washed withice cold methanol (20 mL) and air dried to leave a cream colored powder(12.3 g, 85%). ¹H-NMR (300 MHz, CDCl₃): δ 8.76 (bs, 1H), 8.57 (d, J=5.3Hz, 2H), 7.98 (dd, J=8.5 Hz, J=1.8 Hz, 1H), 7.95 (s, 1H), 7.85 (d, J=8.5Hz, 1H), 7.86-7.76 (m, 2H), 7.55-7.46 (m, 2H), 7.21 (dd, J=4.4 Hz, J=1.2Hz, 2H), 4.68 (d, J=6.5 Hz, 1H), 3.10 (dd, J=17.0 Hz, J=7.7 Hz, 1H),2.90 (dd, J=17.0 Hz, J=1.8 Hz, 1H); ESI MS (M+H)⁺=302; HPLC method AR_(t)=3.11 minutes.

6-(Naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3(2H)-one (10-6): A roundbottom flask equipped with a stir bar was charged6-(naphthalen-2-yl)-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one(10-5, 12 g, 40 mmol), water (6 mL) and DMSO (250 mL). The mixture wasstirred and treated with N-bromosuccinimide (35 g, 200 mmol). Thereaction mixture turned yellow and a slight exotherm was noted while allsolid contents went into solution. After stirring for 20 hours themixture was poured into a stirring solution of water (1000 mL) andsaturated sodium bicarbonate solution (500 mL) at such a rate thateffervescence remained under control. The white precipitate that formedwas collected on a glass frit, washed with water (2×50 mL) and air driedto leave 11.8 g (98%). ¹H-NMR (300 MHz, CDCl₃): δ 11.13 (bs, 1H), 8.55(bs, 2H), 7.80 (dd, J=9.1 Hz, J=2.1 Hz, 1H), 7.76-7.68 (m, 3H),7.55-7.45 (m, 2H), 7.16 (dd, J=8.8 Hz, J=2.0 Hz, 1H), 7.09 (d, J=5.9 Hz,2H), 7.05 (s, 1H); ESI MS (M+H)⁺=300; HPLC method A R_(t)=2.88 minutes.

6-Chloro-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine: (10-7): In around bottom flask equipped with a stir bar was charged with6-(Naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3(2H)-one (10-6, 11.7 g,39 mmol), tetraethyl ammonium chloride (9.42 g, 39 mmol),diisopropylethylamine (6.9 mL) and POCl₃ (167 g). The resulting mixturewas heated at 100° C. for ˜3 hr (reaction monitored by HPLC). Thereaction mixture was cooled to ambient temperature and concentratedunder reduced pressure. The remaining POCl₃ was azeotropically removedusing toluene. The reaction mixture was cooled in an ice bath, treatedwith ice (2 g) and stirred for an hour. The mixture was neutralized withsaturated sodium carbonate to a pH=9 followed by extraction with ethylacetate. The combined organic layer was separated, washed with brinedried (Na₂SO₄) and evaporated. The product was purified bychromatography on silica gel eluted with ethyl acetate in hexanes (1:1to 2:1). The purified product 10-7 was isolated as an off-white solid(9.7 g) 78% yield. ¹H-NMR (300 MHz, CDCl₃) δ 8.59 (dd, 2H, J=4.3 Hz,J=1.8 Hz), 8.03 (d, J=1.4 Hz, 1H), 7.85-7.65 (m, 3H), 7.72 (s, 1H),7.58-7.47 (m, 2H), 7.33 (dd, 1H, J=8.5 Hz, J=1.8 Hz), 7.15 (dd, 2H,J=4.7 Hz, J=1.8); Mass m/z=317.

6-(4-methylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine:(27): (MW-150): A solution of6-chloro-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (10-7) (9.0 g,28.3 mmol) in ethanol (150 mL) in a 500 mL round bottom flask wastreated with 1-methylpiperazine (14.4 g, 141 mmol) and heated to refluxfor 18 h. The solvent was evaporated in vacuo. The residue was dissolvedin ethyl acetate (300 mL) and washed with water (2×200 mL) and saturatedsodium bicarbonate solution (100 mL). The product precipitated from theorganic layer and was collected via filtration on fritted glass. Theproduct was washed with water (20 mL) and ethyl acetate (50 mL) anddried under high vacuum to leave 8.6 g (90%) of 27 as light yellowsolid. ¹H-NMR (300 MHz, CDCl₃) δ 8.54 (dd, 2H, J=4.4 Hz, J=1.7 Hz), 7.94(s, 1H), 7.82-7.68 (m, 3H), 7.51-7.42 (m, 2H), 7.35 (dd, 1H, J=8.5 Hz,J=1.8 Hz), 7.15 (dd, 2H, J=4.4 Hz, J=1.7), 6.88 (s, 1H), 3.82 (dd, 4H,J=5.2 Hz, J=5.0 Hz), 2.60 (dd, 4H, J=5.2 Hz, J=5.0 Hz), 2.39 (s, 3H);ESI MS (M+H)⁺=382; HPLC method A R_(t)=2.58 minutes.

Formation of salt forms of the base to hydrochloride hydrates:

6-(4-Methylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazinedihydrochloride dihydrate (42): In a round bottom flask fitted withcondenser and dry tube, compound6-(4-methylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(27) (7.75 g, 20.3 mmol) was suspended in (˜70 mL) anhydrous isopropanol(99.5%, Aldrich) and heated to 87° C. with stirring until dissolved. Tothe resulting solution 2.5 equiv (4.31 uL) of ultrapure HCl (12N, JTBaker Ultrex® II, Product 6900-05) was added in-portion inducingformation of solids in suspension. The resulting solution was stirred at80° C. for 10 min, cooled to ambient temperature and placed on ice for2.5 h. The suspension was then transferred to 4° C. for an additional 10hr. The resulting yellow precipitate was filtered on a medium fritsintered glass funnel using house vacuum, immediately washed with (3×35mL) of ice-cold anhydrous isopropanol followed by (3×50 mL) of ice-coldanhydrous ether and air dried by house vacuum for 7 h. The product 42was then dried in a glass desiccator containing silica gel in vacuountil the compound gave a constant weight. Product 42 obtained as ayellow powder 90% (gravimetric) yield, with HPLC purity 98% (HPLC methodA); ESI m/z (MeOH): 382.2 (MH⁺); ¹H NMR (300 MHz, CDCl₃): δ 8.55 (dd,J=1.55, 4.45 Hz, 2H); 7.94 (s, 1H); 7.80-7.69 (m, 3H); 7.49-7.34 (m,3H); 7.16 (dd, J=1.5, 4.5 Hz, 2H); 6.88 (s, 1H); 3.83 (t, J=4.8, 4.8,4H); 2.62 (t, J=4.9, 4.9, 4H); 2.39 (s, 3H); HRMS 381.1959 (calculatedfor C₂₄H₂₃N₅ 381.1953).

MW-150 hydrochloride hydrate: (42): EA calculated for C₂₄H₂₉Cl₂N₅O₂: C,58.78; H, 5.96; N, 14.28; Cl, 14.46; O, 6.52; experimentally found C,57.88; H, 5.72; N, 14.03; Cl, 14.31; O, 1.84.

Example 10-1

Alternatively, MW-150 hydrochloride hydrate (42) can be prepared asdescribed below.

2-naphthoyl chloride: In a 1 L round bottom flask was charged with2-naphthoic acid (50 g, 290 mmol) and SOCl₂ (375 mL). The solution wasrefluxed for 4 hours and then concentrated to yield yellow oil, whichwas used without further purification (55.28 g, 100% yield).

N-methoxy-N-methyl-2-naphthamide (10-2): A mixture of 2-naphthoylchloride (1 eq, 35 g, 0.18 mol) and N,O-dimethylhydroxylaminehydrochloride (1.2 eq, 21.5 g, 0.22 mol) in dichloro-methane (1 L) wasstirred, cooled in an ice bath and treated with diisopropylethylamine(2.5 eq, 58 g, 0.45 mol, 81 mL) drop wise over 30 min. The mixture waswarmed to 20° C. over 2 h then stirred an additional lh. The solventswere evaporated and the residual solid was dissolved in ethyl acetate(500 mL) and water (500 mL). The organic layer was separated and washedwith 1N HCl (300 mL), water (300 mL) and brine (200 mL). The organicphase was dried (Na₂SO₄) and evaporated to leave the product as lighttan oil (10-2, 39.5 g, 100%), mass: m/z 216 (M+H)⁺.

1-(Naphthalen-2-yl)-2-(pyridin-4-yl)ethan-1-one (10-3): A solution of4-picoline (1 eq, 15.6 g, 168 mmol, 16.2 mL) in anhydrous THF (1 L)under nitrogen atmosphere at −78° C. was treated with a solution offreshly prepared LDA [from a solution of diisopropyl amine (25.4 g, 252mmol, 35.4 mL)] in THF (140 mL) under nitrogen atmosphere in an ice bathtreated with n-butyllithium solution (2.5M in hexanes, 100 mL, 252 mmol)and stirred for 30 min) over 30 min via cannula. The mixture was stirredat −78° C. for lh and was treated with N-methoxy-N-methyl-2-naphthamide(10-2), (1.1 eq, 40 g, 186 mmol) in THF (160 mL) drop wise over 1.5 hwhile ensuring the temperature was maintained at or below −75° C. Themixture was then allowed to warm to 20° C. over 4 h and stirred anadditional 16 h at this temperature. Saturated ammonium chloridesolution (100 mL) was added to the mixture and allowed to stir for 30min. The solvents were evaporated in vacuo to approximately 20% of theoriginal reaction mixture volume and the residue was dissolved in ethylacetate (1 L) and water (600 mL). The organic layer was separated andwashed with water (600 mL). The product was extracted from the organiclayer with 1N HCl (2×300 mL). The combined acid extracts wereneutralized with solid sodium bicarbonate to pH=8. The precipitatedproduct was filtered, washed with water (2×50 mL) and dried under vacuumfor 16 h to leave a light yellow solid 10-3 (34.5 g, 83%). ¹H NMR (300MHz, CDCl₃) δ 8.58 (dd, J=4.4, 1.8 Hz, 2H), 8.53 (s, 1H), 8.05 (dd,J=8.5, 1.8 Hz, 1H), 8.00-7.84 (m, 3H), 7.66-7.55 (m, 2H), 7.25 (m, 2H),4.43 (s, 2H); Mass (ESI) m/z 248 (M+H)⁺.

Ethyl 4-(naphthalen-2-yl)-4-oxo-3-(pyridin-4-yl)butanoate (10-4): In around-bottom flask equipped with stir bar, an addition funnel and athermometer was added sodium hydride (60% in mineral oil, 0.930 g, 23.3mmol, 1.15 eq) and anhydrous THF (60 ml) under argon atmosphere. Themixture was stirred for 30 min at −5 to −10° C. A solution of1-(Naphthalen-2-yl)-2-(pyridin-4-yl)ethan-1-one (10-3, 5 g, 20.2 mmol,1.15 eq) in anhydrous THF (40 ml, cold) was added via an addition funnelwith continuous stirring while keeping the temperature ˜−5° C. Themixture was stirred for 20-30 min (deep yellow). To this mixture wasadded a solution of ethyl 2-bromoacetate (3.8 g, 23.3 mmol, 1.15 eq) inanhydrous THF (40 ml, cold) with vigorous stirring (color changed backto yellow). The reaction mixture stirred at ambient temperature for 3-4h (reaction progress monitored by HPLC, 68-71% product peak after 2.5 h)and was quenched with crushed ice. The solvents were evaporated atreduced pressure to approximately 80% of the original volume and theresidue was diluted with a mixture of ethyl acetate and water (30 ml,3:2 v/v). To this mixture was added cold saturated NaHCO₃ solution (˜150ml) to neutralize to pH ˜8 and the product was extracted with ethylacetate (2×200 ml). The combined ethyl acetate layers were washed withbrine (2×50 ml), dried over anhydrous Na₂SO₄ and evaporated to a crudesticky residue. Trituration with hexane produced a yellow solid that wascollected on a medium glass frit via suction filtration and dried toleave the product 10-4 (5.1 g, 75%). ¹H NMR (500 MHz, CDCl₃) δ 8.53 (d,J=5.0, 2H), 8.49 (bs, 1H), 7.99 (dd, J=8.6 Hz, J=1.8 Hz, 1H), 7.92 (d,J=8.3 Hz, 1H), 7.86-7.82 (m, 2H), 7.60-7.50 (m, 2H), 7.30 (dd, J=1.7,4.5 Hz, 2H), 5.27 (m, 1H), 4.12 (q, J=7.0 Hz, 2H), 3.42 (dd, J=17.0 Hz,J=9.3 Hz, 1H), 2.80 (dd, J=17.0 Hz, J=5.3 Hz, 1H), 1.21 (t, J=7.1 Hz,3H); ESI MS (MH)⁺=334.04.

6-(Naphthalen-2-yl)-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one(10-5): A solution ethyl4-(naphthalen-2-yl)-4-oxo-3-(pyridin-4-yl)butanoate (10-4) (4 g, 1 eq)in ethanol in a round bottom flask was cooled to below 10° C. and wastreated with hydrazine monohydrate. Reaction mixture was refluxed for˜4-5 h with stirring (until all starting material was consumed,monitored by HPLC). The mixture was cooled to ambient temperature andconcentrated under reduced pressure to remove all solvents to get theyellowish orange semi solids. To the flask was added ˜10 ml methanol andcooled to ˜4° C. (kept in refrigerator for ˜3 h). The white solids thatformed were collected on a medium glass frit via suction filtration,washed with chilled methanol (2×10 ml) and dried under vacuum to obtainthe product 10-5 (3 g, 83%). ¹H NMR (300 MHz, CDCl₃) δ 8.76 (bs, 1H),8.57 (d, J=5.3 Hz, 2H), 7.98 (dd, J=8.5, 1.8 Hz, 1H), 7.95 (s, 1H), 7.85(d, J=8.5 Hz, 1H), 7.86-7.76 (m, 2H), 7.55-7.46 (m, 2H), 7.21 (dd,J=4.4, 1.2 Hz, 2H), 4.68 (d, J=6.5 Hz, 1H), 3.10 (dd, J=17.0, 7.7 Hz,1H), 2.90 (dd, J=17.0, 1.8 Hz, 1H); Mass (ESI) m/z 302 (MH)⁺; LC/MSmethod A R_(t)=3.11 min.

6-(Naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3(2H)-one (10-6): In around-bottom flask equipped with stir bar and condenser with dry tubewas charged6-(Naphthalen-2-yl)-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one(10-5) (3.2 g, 10.6 mmol, 1 eq) in glacial acetic acid. A solution ofbromine (1.78 g, 11.2 mmol, 1.05 eq) in acetic acid was added withvigorous stirring at 70° C. and refluxed until all starting materialdisappeared ˜2.5 h (reaction monitored by HPLC). The reaction mixturewas cooled to ambient temperature, concentrated under vacuum, pouredonto crushed ice and stirred for 1 h. To this reaction mixture cold 10%sodium carbonate solution was added at such a rate that effervescenceremained under control and the pH was adjusted to 9. The whiteprecipitate that formed was collected on a glass frit via suctionfiltration, washed with water twice (3×20 ml) and dried under vacuum toobtain the product 10-6 (3.1 g, 97%). ¹H NMR (300 MHz, CDCl₃) δ 11.13(bs, 1H), 8.55 (bs, 2H), 7.80 (dd, J=9.1, 2.1 Hz, 1H), 7.76-7.68 (m,3H), 7.55-7.45 (m, 2H), 7.16 (dd, J=8.8, 2.0 Hz, 1H), 7.09 (d, J=5.9 Hz,2H), 7.05 (s, 1H); Mass (ESI) m/z 300 (MH)⁺; LC/MS method A ^(t)R=2.88min.

6-Chloro-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (10-7): A roundbottom flask equipped with a stir bar was charged with6-(Naphthalen-2-yl)-5-(pyridin-4-yl)pyridazin-3(2H)-one 10-6 (2.81 g,9.4 mmol, 1 eq), dry acetonitrile (60 ml) and POCl₃ (17.2 g, 11.2 mmol,12 eq). The mixture was heated at 90° C. for ˜3 h (disappearance ofstarting material as monitored by HPLC). The reaction was quenched withaddition of crushed ice followed by removal of acetonitrile underreduced pressure. The mixture was poured onto crushed ice and stirredfor ˜1 h, and neutralization done by addition of cold 10% NaOH (pH ˜9)while stirring. The product precipitated as fine particles which werecollected on a glass frit under vacuum, washed with water (3×25 ml) anddried under vacuum to yield the light beige solid compound 10-7 (2.54 g,85%). ¹H NMR (CDCl₃, 500 MHz) δ 8.66 (bs, 2H), 8.09 (s, 1H), 7.90-7.80(m, 3H,), 7.64 (s, 1H), 7.62-7.55 (m, 2H), 7.39 (d, J=8.5 Hz, 1H), 7.24(bs, 2H). Mass (ESI) m/z=317.94 (MH)⁺.

6-(4-Methylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(27): A solution of6-chloro-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine (10-7) (2.54 g,7.9 mmol, 1 eq) in 95% ethanol (40 ml) was reacted with1-methylpiperazine (4 g, 40 mmol, 5 eq) under reflux for ˜18 h. Aftercooling to ˜20° C., ethanol is evaporated under reduced pressure and theresultant residue is dissolved in ethyl acetate: saturated aqueoussodium bicarbonate (2:1) mixture. The separated organic layer was twicewashed with water (2×) which yielded the precipitated product in theorganic layer. The solid product is harvested on a medium glass fritunder vacuum, washed twice with ethyl acetate, and dried under vacuum(up to 12 h). Product 27 (2.6 g, 90%) was obtained as a pale yellowsolid. ¹H NMR (CDCl₃, 500 MHz) δ 8.53 (dd, J=1.6, 4.4 Hz, 2H), 7.92 (s,1H), 7.78 (dd, J=5, 7.5 Hz, 1H,), 7.71-7.67 (m, 2H), 7.47-7.41 (m, 2H),7.34 (dd, J=5, 7.5 Hz, 1H), 7.13 (dd, J=5, 5 Hz, 2H), 6.86 (s, 1H), 3.83(m, 4H), 2.62 (t, J=5 Hz, 4H), 2.39 (s, 3H). Mass (ESI) m/z 382.20(MH)⁺; LC/MS method R_(t)=2.58 min.

6-(4-Methylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazinedihydrochloride dihydrate (42): In a round-bottom flask fitted withcondenser and dry tube, compound6-(4-methylpiperazin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine(8, 9.1 g, 23.8 mmol) was suspended in 100 mL of anhydrous isopropanol(99.5%, Aldrich) and heated to 81° C. with stirring until dissolved. Tothe resulting solution, ultrapure HCl (12 N, JT Baker Ultrex II, product6900-05) (2.5 equiv, 5.1 mL, 59.63 mmol) was added in-portion, inducingformation of solids in suspension. The resulting solution was stirred at81° C. for 10 min, cooled to ambient temperature, and placed on anice-bath for 2.5 h. The suspension was then stored at 4° C. for anadditional 10 h. The resulting yellow precipitate was filtered on amedium frit sintered glass funnel using a house vacuum, immediatelywashed three times with ice-cold anhydrous isopropanol (35 mL), followedby ice-cold anhydrous ether (50 mL), and air-dried using house vacuumfor 7 h. The product was then dried in a glass desiccator containingsilica gel in vacuo until the compound gave a constant weight. Product42 obtained as a yellow powder (11.12 g, 96%), HPLC purity>98% (LC/MS).ESI m/z=382.2 (MH)⁺. ¹H NMR (500 MHz, CD₃OD) δ 8.75 (dd, J=1.5, 5.0 Hz,2H), 7.96 (s, 1H), 7.91-7.83 (m, 6H), 7.58-7.52 (m, 2H), 7.38 (dd,J=1.8, 8.4 Hz, 1H), 4.86 (m, 2H), 3.71 (m, 2H), 3.57 (m, 2H), 3.28 (m,2H), 3.01 (m, 3H). HRMS (mass) calculated for C₂₄H₂₃N₅: 381.19535.Found: 381.1955. Elemental analysis calculated (%) for C₂₄H₂₉C₁₂N₅O₂: C,58.78; H, 5.96; N, 14.28; Cl, 14.46; O, 6.52. Found: C, 58.89; H, 5.89;N, 14.15; Cl, 14.27; O, 7.05. mp 240° C., decomposes.

Example 10-2

Compound 56(6-(4-methylpiperidin-1-yl)-3-(naphthalen-2-yl)-4-(pyridin-4-yl)pyridazine)(also described herein as SRM-137A) can be prepared in a similar manneras described in Example 10-1 by using 4-methylpiperidine instead of1-methylpiperazine.

Example 10-3

Compound 60(3-(naphthalen-2-yl)-4-(pyridin-4-yl)-6-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine)(also described herein as SRM-203B) can be prepared in a similar manneras described in Example 10-1 by using 2-(piperazin-1-yl)pyrimidineinstead of 1-methylpiperazine.

Example 11

6-chloro-N,N-dimethylpyrazin-2-amine (11-2): A mixture of2,6-dichloro-pyrazine (1.2 g, 13.4 mmol, 1 eq) and 35 mL acetonitrilewas combined in a heavy wall pressure vessel followed by the addition of40% dimethyl amine (14.7 mmol, 1.1 eq) and K₂CO₃ (16.1 mmol, 1.2 eq) andpurged with argon. The pressure vessel was capped and heated at 900° C.for 4 hr (monitored by TLC/HPLC). The reaction mixture was then cooledto ambient temperature, transferred to a round bottom flask andconcentrated in vacuo. The residue was treated with 20 mL of Milli-Qwater and the aqueous layer was extracted several times withdichloromethane using a separatory funnel. The combined organic layerswere dried with anhydrous sodium sulfate and concentrated in vacuo. Theproduct 2 was purified by column chromatography on silica gel (200-400mesh) using ethyl acetate: hexane (1:1 v/v) eluent. Product 11-2 wasobtained as white solid (1.1 g) in 60% (gravimetric) yield, with anapparent HPLC (tr/purity): 18.4 min, >90% (HPLC method A); (ESI) m/z(MeOH): 158.10 (MH⁺).

N,N-dimethyl-6-(pyridin-4-yl)pyrazin-2-amine (11-3): A mixture of6-chloro-N,N-dimethylpyrazin-2-amine (11-2) (1 g, 6.3 mmol) andpyridin-4-yl boronic acid (1.06 g, 8.6 mmol, 1.35 eq) were suspended indimethoxyethane and water (10:1) in a heavy wall pressure vessel. Thereaction mixture was purged with argon for 10 min. Sodium carbonate (2.0g, 19.6 mmol, 3 eq) and tetrakis(triphenylphosphine) palladium (0.650 g,0.57 mmol) were added and the reaction mixture was heated at 110° C. for15 hrs. The reaction mixture was cooled to ambient temperature andfiltered through a medium frit sintered glass funnel filled with Celite.The filtrate was concentrated under reduced pressure, dissolved indichloromethane and washed with water (3×5 mL). The organic layer wasdried with sodium sulfate and concentrated under reduced pressure. Theproduct 11-3 was purified over silica gel column using ethyl acetate andhexane (2:3 v/v) eluent. The title compound 11-3 was obtained as yellowsolid (1.1 g) in 75% (gravimetric) yield. HPLC (tr/purity): 10.7min, >90% (HPLC method A); ESI m/z (MeOH): 201.10 (MW).

5-bromo-N,N-dimethyl-6-(pyridin-4-yl)pyrazin-2-amine (11-4): Bromine(282 μL, 54.9 mmol, 1.1 eq) was added dropwise to the solution ofN,N-dimethyl-6-(pyridin-4-yl)pyrazin-2-amine (11-3) (1 g, 50 mmol) in 5mL of acetic acid at room temperature with stirring. The reactionprogress was monitored by HPLC until all starting material disappeared(˜4 hr). The reaction mixture was poured onto crushed ice and treatedwith (5%) sodium bisulfate solution to remove the excess bromine. The pHof the aqueous reaction mixture was adjusted to pH 7 with 0.2 N NaOH andthe mixture was extracted several times with dichloromethane. Thecombined organic phase was dried over Na₂SO₄ (anhydrous) andconcentrated under reduced pressure. Product 4 was purified by silicaget (200-400 mesh) column chromatography using dichloromethane and 5%MeOH as eluent, Product 11-4 was obtained as pinkish solid (600 mg) in76% (gravimetric) yield. HPLC (tr/purity): 13.80 min, >90% (HPLC methodA); (ESI) m/z (MeOH): 279.10 (MH⁺).

N,N-dimethyl-5-(naphthalen-1-yl)-6-(pyridin-4-yl)pyrazin-2-amine (44):(MW-064): A mixture of5-bromo-N,N-dimethyl-6-(pyridin-4-yl)pyrazin-2-amine (11-4) (0.500 g,1.7 mmol) and 1-napthylboronic acid (0.422 g, 2.4 mmol) were suspendedin dimethoxyethane (DME) and water (10:1) in a heavy wall pressurevessel. The reaction mixture was purged with argon for 10 min.Subsequently tetrakis(triphenylphosphine) palladium (0.186 g, 9 mol %)and sodium carbonate (0.588 g, 5.5 mmol) were added and the reactionmixture was heated at 110° C. for 15 hrs. The reaction mixture wascooled to ambient temperature and filtered through a medium fritsintered glass funnel filled with Celite. The filtrate was concentratedunder reduced pressure and added ˜15 mL MilliQ water and extractedseveral times in dichloromethane. The combined organic layers was driedover anhydrous sodium sulfate and then concentrated under reducedpressure. The product 44 was purified by column chromatography on silicagel (200-400 mesh) using ethyl acetate and hexane (2:3 v/v) eluent. Theproduct 44 was obtained as pale yellow powder (320 mg) in 45%(gravimetric) yield. ¹H NMR (500 MHz, CDCl₃): δ 8.34 (b, 2H); 8.23 (s,1H); 7.88 (dd, J=8.4 Hz, 2H); 7.74 (d, J=8.4 Hz, 1H); 7.48-7.36 (m, 3H);7.26-7.22 (m, 3H); 3.29 (s, 6H). HPLC (tr/purity): 16.3 min>97% (methodA); mass (ESI) of m/z (CD₃OD): 327.20 (MH⁺); HRMS calculated forC₂₁H₁₈N₄ 326.1531 found 326.1532.

Example 11-1

Compound 63(5-(4-methylpiperazin-1-yl)-2-(naphthalen-2-yl)-3-(pyridin-4-yl)pyrazine)(also described herein as SRM-138B) can be prepared in a similar manneras described in Example 11 by using 1-methylpiperazine instead ofdimethyl amine and by using 2-naphthylboronic acid instead of1-naphthylboronic acid.

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D. Martin Watterson, Valerie L. Gram-Tokars, Saktimayee M. Roy, James P.Schavocky, Brinda D. Bradaric, Adam D. Bachstetter, Bin Xing, EdgardoDimayuga, Faisal Saeed, Hong Zhang, Agnieszka StanisZewski, Jeffrey C.Pelletier, George Minasov, Wayne F. Anderson, Ottavio Arancio, Linda J.Van Eldic, PLOS ONE, 2013, 8, e66226.

Munoz L, Ralay Ranaivo H, Roy S M, Hu W, Craft J M, McNamara L K, WingChico L, Van Eldik L, and Watterson D M; A novel p38α MAPK inhibitorsuppresses brain proinflammatory cytokine up-regulation and attenuatessynaptic dysfunction and behavioral deficits in an Alzheimer's diseasemouse model. J Neuroinflammation 2007, 4: 21.

Hu W, Ralay Ranaivo H, Roy S M, Behanna H A, Wing L K, Munoz L, Guo L,Van Eldik L J, Watterson DM; Development of a novel therapeuticsuppressor of brain proinflammatory cytokine up-regulation thatattenuates synaptic dysfunction and behavioral deficits. BioorgMed ChemLett 2007, 17: 414-418.

Schmitt M, de Araújo-Júnior J X, Oumouch S, Bourguignon J J; Use of4-bromo pyridazine 3,6-dione for building 3-amino pyridazine libraries.Molecular Diversity, 2006, 10: 429-434

Tamayo N, Liao L, Goldberg M, Powers D, Tudor Y, Yu V, Wong Lu Henkle B,Middleton, S, Syed R; Design and synthesis of potent pyridazineinhibitors of p38 MAP kinase. Bioorg Med Chem Lett 2005, 15: 2409-2413.

Example 12: Kinase Assays

The concentration dependent ability of compounds to inhibit human p38αMAPK, p38β MAPK and CK-1δ were done essentially as described in J.Neurosci. 2012, 32, 10201 (herein incorporated by reference in itsentirety) and by use of commercially available Millipore® Drug Discoveryand Development services. IC₅₀ values were calculated by generating a10-11 point curve and analyzed using nonlinear regression curve fit inGraphPad Prism statistical software. Z-factor (≥0.5) calculation and %CV (<20%) were calculated for each assay. Large-scale kinome screenswere done using the commercially available Millipore® Profiler testsystems (www.millipore.com) that included >291 protein kinasesrepresentative of all major kinome branches as well as isoforms ofindividual families. The NCBI Entrez identifier for each kinase isprovided at the vendor site. A hierarchal analysis was done oninhibitors. First, an initial screen was done where each inhibitor wastested at a fixed concentration (20,000 nM) against a panel of proteinkinase targets covering the major branches of the kinome, using an ATPconcentration for each kinase at or near their apparent Km. Second,preliminary hits from the profiler screen were validated by a follow-upconcentration dependent test of the inhibitor to obtain an IC₅₀ valuefor the inhibitor and a given kinase in order to confirm the hit aspositive. Third, kinetic analyses to determine a Ki value were done onconfirmed positives with IC₅₀ values<1,000 nM.

The commercially available europium competitive active site bindingassay (LanthaScreen® Eu Kinase Binding Assay, Invitrogen Life ScienceTechnologies, Grand Island, N.Y., USA) that is based on time-resolvedfluorescence resonance energy transfer (TR-FRET) technology was used totest for competitive binding to the non-activated form of p38α MAPK.IC₅₀ values were generated from triplicate 12-point curves. Assays wereperformed in a 384 well plate format (Corning cat No. 3673) in a finalvolume of 15 μL containing 5 nM kinase (Invitrogen cat No. PV3305), 5 nMtracer (Invitrogen cat No. PV5830), 2 nM antibody (Invitrogen cat No.PV5594). Plates were centrifuged (240 g, 5 min) to mix ingredients,incubation done for 60 min at 25° C., and plates centrifuged (240 g, 5min) to concentrate reaction mixtures in the plate bottom well. Readingswere taken in an En Vision Plate Reader (Perkin Elmer; Waltham Mass.,USA) using a dichroic mirror and excitation at wavelength 340 nm (30 nmbandpass) and emission at 665 nm (10 nm bandpass) and 615 nm (10 nmbandpass). Delay time was 100 μs, and integration time 200 μs. Theemission ratio was determined by dividing the acceptor/tracer emission(665 nM) by the antibody/donor emission (615 nM). Data were expressed aspercent of the maximal binding activity, as determined by the emissionratio, and IC₅₀ values were calculated using GraphPad Prism, version5.0a, by a nonlinear regression data analysis of log inhibitorconcentration versus emission ratio.

Off-Target Functional Screens

Compounds were screened in vivo using a standard dose escalation screenin mice coupled with clinical observation and testing based on theSHIRPA mouse phenotype assay paradigm (Mammalian Genome 8, 711-719SHIRPA (1997); herein incorporated by reference in its entirety). Toobtain insight into the potential for off-target activity with thelargest known family of small molecule drug targets, G-protein coupledreceptors (GPCRs), a cell-based functional screen of the final titlecompounds was done. The Millipore® GPCR Profiler screen employs theChemiScreen GPCR stable cell line technology used for real-time calciumflux FLIPR assays on a panel of 158 GPCRs. The NCBI Entrez identifierfor each GPCR is provided at the vendor site (www.millipore.com).Follow-up validation of any initial hits as being true positives ornegatives was done as described above for protein kinases by testing theconcentration dependence of any cellular effects. No IC₅₀ values<1,000nM were detected.

p38α MAPK activity retention and improved no adverse effect levels inmice were observed with simplification of the amine. Surprisingly,introduction of bulkier groups, for example naphthyl, at the R3 positionof the pyridazine provided improved occupancy at the hydrophobic pocketof the enzyme. The introduction of large groups at this positionsurprisingly improved p38α MAPK affinity and reduced affinity for Ck1δ(Table 2).

TABLE 2 Target Family Screen: p38α MAPK p38α MAPK Ck1δ Cmpd IC₅₀(nM)K_(i)(nM) IC₅₀ (nM)  1 (MW-181) 215 184 >10,000  2 (MW-108) 236 1146,118  3 (MW-066)  481 ± 174 130 ± 40  4 (MW-177) 238 ± 49 369 ± 30  5(MW-207) 408 ± 41 284 ± 30  6 (MW-105) 543 ± 29 657 843 ± 42  7 (MW-077)419 186 >10,000  9 (MW-125) 412 127 >10,000 10 (MW-167) 307 86 >10,00011 (MW-122) 185 98 >10,000 12 (MW-124) 191 >10,000 13 (MW-107) 26491 >10,000 14 (MW-109) 131 >10,000 15 (MW-156) 289 276 >10,000 16(MW-200) 175 100 4,984 17 (MW-078) 1775 >10,000 18 (MW-085) 1166 19(MW-155) >1600 20 (MW-082) >3200 >10,000 21 (MW-165) >1600 22 (MW-066)369 4,283 23 (MW-033) 162 >10,000 24 (MW-010) 188 >10,000 25 (MW-031)162 >10,000 26 (MW-025) 130 2,620 27 (MW-150) 282 101 2,802 28 (MW-118)518 403 29 (MW-108) 3070 6126 30 (MW-126) 753 2628 31 (MW-146) 240 6,45832 (MW-148) 185 2,989 33 (MW-152) 481 34 (MW-154) 112 2,162 35 (MW-153)189 36 (MW-164) 224 101 11,227 37 (MW-149) 235 4,619 44 (MW-064) 456343 >10,000 49 (MW-203) 1176 6,352 50 (MW-017) 974 16,752 51 (MW-044)2096 39,869 52 (MW-032) 735 17,897 53 (MW-059) >20,000 118,989 54(MW-197) 669 135,537 55 (MW-063) 526 32,522 61 (MW-086) >20,000 9,087 62(MW-026) 520 4,993

Example 13: Cell-Based Assays

Quantitative Cell-Based Activity Screen

The ability of compounds to reduce stressor-induced up-regulation ofproinflammatory cytokine production was tested in the murine microglialBV-2 cell line stimulated with LPS as previously described Neuroinfl.2011, 8, 79; herein incorporated by reference in its entirety). Forexperiments, cells were plated in 48-well tissue culture plates at 2×10⁴cells/well and cultured for 24 hrs. Serum-containing medium was thenremoved and cells were treated with either saline vehicle control or 100ng/ml LPS stimulus (LPS from Salmonella enterica serotype typhimurium;EU/mg 600,000; Sigma L6143) in the absence or presence of increasingconcentrations of compound, with at least six concentrations of compoundranging from 0.45 μM to 30 μM. Stock solutions of compounds were made insterile saline (0.9% sodium chloride) that was free of preservatives(Hospira, Inc., Lake Forest, Ill.: NDC 0409-4888-10). Solutions for celltreatments were prepared by dilution of the stock solutions intoserum-free media immediately before adding to the cells. Compound wasadded to cell cultures just before LPS addition. In some experiments,BV-2 cells were stimulated with other TLR ligands as describedpreviously (J. Neuroinfl. 2011, 8, 79; herein incorporated by referencein its entirety). TLR ligands used were TLR2: 10 μg/ml LTA, TLR4: 100ng/ml LPS, TLR7/8: 500 ng/ml CL097, and TLR9: 500 ng/ml ODN1668. Cellswere harvested after 18 hrs of treatment for cytokine measurements.

Levels of IL-1β in cell lysates were measured by ELISA using kits fromMeso Scale Discovery (MSD; Gaithersburg, Md.), as previously described(J. Neuroinfl. 2011, 8, 79; herein incorporated by reference in itsentirety). For the most consistent cytokine measurements with the lowestintra- and interassay variability, cell lysates were frozen at −80° C.for at least one hour, then thawed prior to cytokine assay. Cytokinelevels were determined by comparison to standard curves ranging from 2.4pg/mL to 10,000 pg/mL, and confirmation that sample values were on thelinear part of the standard curves. The levels of IL-1β were normalizedto the LPS-stimulated vehicle-treated control group for each 48-welltissue culture plate, with data presented as percent of LPS alone. Datarepresent 3-to-10 independent experiments.

Cell-Based Target Engagement and Activity

The concentration dependent ability of the compounds to inhibitLPS-induced phosphorylation of MK-2, a highly selective p38α MAPKsubstrate, was examined in BV-2 cells as previously described (J.Neuroinfl. 2011, 8, 79; herein incorporated by reference in itsentirety). BV-2 cells were plated in 48-well tissue culture plates at2×10⁴ cells/well and treated in serum containing media with LPS in theabsence or presence of compounds as described above. After 60 min oftreatment, cell lysates were prepared, and phospho-MK-2 levels weremeasured by MSD ELISA kits. MSD signal was baseline corrected bysubtracting the unstimulated vehicle-treated control group. The datawere then normalized to the LPS-stimulated vehicle-treated control groupfor each 48-well tissue culture plate. Data are presented as percent ofLPS alone, and represent 2-to-3 independent experiments.

The loss of inhibitory activity in LPS-induced microglia where theendogenous p38α MAPK gene was disrupted by knock-in of a fully activebut drug resistant MAPK mutant, p38αT106M knock-in (J. Biol. Chem. 2007,282, 34663; herein incorporated by reference in its entirety), wastested in primary microglia cultures as previously described (J.Neuroinfl. 2011, 8, 79; herein incorporated by reference in itsentirety). Mixed glial cultures were prepared from the cerebral cortexof 1-3 day old neonatal C57Bl/6 mice (WT) or p38αT106M knock-in mice,and microglia were isolated from the mixed glial cultures by theshake-off procedure as described (J. Neuroinfl. 2011, 8, 79; hereinincorporated by reference in its entirety). Microglia were stimulatedwith LPS (3 ng/mL) or vehicle control for 24 hrs in the absence orpresence of inhibitors, and IL-1β levels measured as described above.Data are expressed as a percent of the maximal activity, where activityin the presence of LPS alone is taken as 100%. Data represent 3independent experiments.

Example 14: In Vivo Efficacy Related to CNS Inflammatory Response

The ability of compounds to inhibit an acute CNS inflammatory responsewas tested in mice administered LPS to induce brain cytokine productionas previously described (J. Neuroinfl. 2011, 8, 79; herein incorporatedby reference in its entirety). 2-month old, female, C57Bl/6 mice wereadministered LPS (0.5 mg/kg) by i.p. injection and received compound (20mg/Kg) or saline vehicle by oral gavage in a volume of 200 μL one hrprior to the LPS injection. At 6 hrs after LPS administration, mice wereeuthanized, perfused with phosphate-buffered saline (PBS), and braincortex homogenate supernatants prepared as described (J. Neuroinfl.2011, 8, 79; herein incorporated by reference in its entirety). IL-1βlevels in 50 μL of supernatant were determined by MSD ELISA. BCA ProteinAssay (Pierce) was used to normalize the total amount of protein in thesample loaded.

Target engagement and validation screens for the glia-based p38α MAPKmediated stress responses that include upregulation of proinflammatorycytokine (PIC) production. These results are summarized in FIG. 1A-1C.The concentration dependent inhibition of increased PIC production overthe same range in which phosphorylation of selective downstreamsubstrates is inhibited in a standard mouse glia cell line (BV2). FIG.1D shows lack of responsiveness to treatment in drug resistant cellsfrom mutant mice in which the endogenous p38α MAPK gene is disrupted byinsertion of a transgene encoding a fully active mutant p38α MAPK wherethe gatekeeper amino acid for the hydrophobic pocket is mutated fromsmall to bulky side chain (T to M), blocking access to the pocket. Thebulkier side chain prevents binding of the inhibitors as a keycharacteristic of target recognition and affinity is entry of theligand's aromatic group into the hydrophobic pocket of the kinase, whichis allowed when a small side chain amino acid is at the gatekeeper butnot when a bulky group is present. As a result, the knock-in mice have ap38α MAPK with normal catalytic activity but are resistant to smallmolecule inhibitors that employ this ligand-target recognition feature.This mechanistic linkage between target engagement in stressed cellmodulation related to the disease progression mechanism is a link amonginhibitors with good in vitro activity and pharmacokinetic properties.FIG. 1E shows in vivo inhibition of LPS induced PIC increase in thecortex. FIG. 1F shows efficacy with extended administration to atransgenic mouse AD model in which the pathology progress is prolongedand involves early PIC increases.

The ability of MW-108 to engage its target in a cellular assay was doneby examining the concentration-dependent inhibition of phosphorylatedMK-2, considered a highly selective p38α MAPK substrate. FIG. 2A showsthe results with the microglial cell line BV-2 stimulated with thestandard glial activating stimulus lipopolysaccharide (LPS). There is aclear decrease in phosphorylated MK-2 in LPS-stimulated cells withincreasing concentration of MW-108 (2). LPS activation of the signaltransduction pathway involving p38α MAPK and its substrate MK-2 resultsin a downstream increase in proinflammatory cytokine production. Levelsof the proinflammatory cytokine interleukin 1β (IL-1β) as a function ofMW-108 (2) concentration (FIG. 2B) were quantified, and showed acoincident decrease over a similar concentration range. The IC₅₀ usinginhibition of IL-1β overproduction as an end point was 610±20 nM.

To extend the general biological relevance of the initial cellularengagement studies, the ability of MW-108 (2) to inhibit increases inIL-1β levels using different stressors, wild-type and inhibitorresistant primary cells, and in vivo responses was examined. As shown inFIG. 2C, the inhibition of IL-1β production is not limited to LPS as astressor, but was also seen in BV-2 cells stimulated with diverse TLRligands (TLR2, TLR4, TLR7/8, TLR9). As shown in FIG. 2D, furthervalidation was provided by demonstrating a loss of MW-108 (2) activityin primary microglia cultures where the endogenous p38α MAPK was removedby targeted knock-in replacement with a fully active but drug resistantmutant p38α T106M MAPK. The knock-in yields catalytically normal kinase,but the replacement of the gatekeeper Thr at residue 106 with a largerside chain Met amino acid renders the mutant p38α T106M MAPK resistantto inhibitors such as MW-108 (2) that exploit the use of the hydrophobicpocket with bulky substituents. To probe the in vivo relevance of theproinflammatory cytokine modulation seen in glia cultures, we tested iforal administration of MW-108 (2) could attenuate stressor induced IL-1βincreases in the brain. As shown in FIG. 2E, oral administration ofMW-108 (2) restored IL-1β levels in the brain cortex back towardscontrol. Altogether, these results demonstrate that MW-108 engages itsmolecular target p38α MAPK and modulates the linked cellular response ofincreased proinflammatory cytokine production, and that thistarget-related function is evident in vivo.

A prevailing hypothesis in kinase inhibitor development based onanalysis of successful campaigns is that in vivo efficacious kinaseinhibitors engage a close set of multiple kinases (Methods Mol. Biol.2012, 795, 1; herein incorporated by reference in its entirety).Relatedly, many existing p38α MAPK inhibitors are equivalent or betterinhibitors of p38β MAPK, NLK, CK1 and other kinases.

To address whether a mixed p38 MAPK inhibitor that lacks CK1 inhibitionwould have cellular activities similar to MW-108 (2), the effects ofMW-181(1) were examined in the same series of target engagement andmechanism of action studies. MW-181 (1) exhibited activities similar toMW-108 (2), including inhibition of increased cytokine production byglia in response to LPS (IC₅₀=820±30 nM) and other TLR ligands,inhibition of MK-2 phosphorylation, loss of activity in drug resistantmicroglia, and in vivo suppression of increased brain levels of IL-1βafter oral administration (FIG. 3). The data indicate that the activityand function of the p38 MAPK family inhibitor, MW-181 (1), reflects itsp38α MAPK inhibition in these assays.

Stressor induced responses include activation of endogenous p38α MAPKwhich, in turn, increases its phosphorylation of endogenous substratessuch as pMK2 and pMSK1. This endogenous kinase activity is the proximalstep in the in vivo mechanism that yields injurious increases inproinflammatory cytokine production by glia. The phosphorylation state(activation) of the p38α substrates pMK2 (FIG. 4A) and pMSK1 (FIG. 4B)are increased at 1 hr after LPS addition and this increase is attenuatedin a dose dependent manner by the inhibitor. Data are expressed aspercent of maximal activity (activity with LPS+vehicle), and arerepresentative of at least 2 independent experiments. The log IC50values and IC50 95% confidence intervals are shown in gray boxes withinthe graphs.

Concentration-dependent suppression of glia transformation viainhibition of endogenous p38α MAPK phosphorylation of endogenous gliasubstrates was examined via serial dilutions of MW-181(1) added to gliastimulated with LPS. At 18 h after LPS addition, IL-1β protein levels incell lysates were measured via ELISA (FIG. 4C).

MW-181 (1) reduces brain cytokine levels in stressed wild type (WT) micebut not in drug resistant strains (KI) that have normal p38α MAPKactivity but cannot bind the inhibitors due to a designed point mutationthat prevents active site binding of the inhibitor. (FIGS. 5A and 5B).

FIG. 6 shows target engagement by MW-150 (27) in glia response pathway.The phosphorylation state (activation) of the p38α substrate pMK2 isincreased at 1 hr after LPS addition and this increase is attenuated ina dose dependent manner by MW-150 (27) (FIG. 6A). IL-1β protein levelsin cell lysates at 18 hrs after LPS addition in MSD ELISA measurementswith vehicle, LPS+vehicle and LPS+MW-150 (FIG. 6B). TNFa protein levelsin conditioned media at 18 hrs after LPS addition in MSD ELISAmeasurements with vehicle, LPS+vehicle and LPS+MW-150 (FIG. 6C). Dataare presented as percent of maximal activity (activity with LPS+vehicle)and are representative of at least two independent experiments.

The concentration dependent engagement of endogenous target and itsproximal endogenous substrate in living cells, and the concentrationdependent effect on upregulated proinflammatory cytokine production instressed cells provides a strong linkage between in vitro kinaseinhibitor activity, pharmacodynamics effects relevant to diversediseases and the efficacy in biological studies of the compounds.

Example 15: In Vivo Models for Alzheimer's Disease

Animal models: Two mouse models of the disease were used. In one model,mice overexpressing the mutated transgenes APP and PS1 (termed APP/PS1mice) were used (Ann. Neurol. 2004, 55, 801; herein incorporated byreference in its entirety) and their WT littermates. APP/PS1 mice arewell characterized with respect to AD pathology, and begin to showsynaptic and memory impairment as early as 3 months of age (Ann. Neurol.2004, 55, 801; Nat. Med. 1998, 4, 97; Exp. Neurol. 2001, 171, 59;Neurobiol. Dis. 1999, 6, 231; and Neurochem. Res. 2003, 28, 1009; eachherein incorporated by reference in its entirety). In the second modelmice that are acutely treated with amyloid-beta are used. APP/PS1 miceare obtained by crossing APP mice with PS1 mice from a breeding colonykept at the animal facility of Columbia University. Animals aregenotyped by PCR as described in Ann. Neurol. 2004, 55, 801; Science1996, 274, 99 (each herein incorporated by reference in its entirety).All of the mice are maintained on a 12 h light/dark cycle (with lightson at 6:00 A.M.) in temperature- and humidity-controlled rooms. Food andwater are available ad libitum. Animals are sacrificed by cervicaldislocation followed by decapitation.

Aβ preparation. Oligomeric Aβ₄₂ is prepared from a commerciallyavailable Aβ₄₂ synthetic peptide (American Peptide Co, Sunnyvale,Calif.), as previously described in J. Biol. Chem. 2003, 278, 11612; J.Neurosci. 2008, 28, 14537 (each herein incorporated by reference in itsentirety). The lyophilized peptide is resuspended in cold1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) and aliquoted inpolypropylene vials. After 24 hrs, the HFIP solution is allowed toevaporate in a fume hood until a thin film of peptide is formed on thebottom of the vials. Peptide films is dried and stored in sealed vialsat −20° C. Prior to use, anhydrous DMSO (Sigma) is added to obtain apure monomeric Aβ/DMSO solution and is sonicated for 10 min (J. Biol.Chem. 2003, 278, 11612; herein incorporated by reference in itsentirety). Oligomeric Aβ₄₂ is obtained by incubating an aliquot ofmonomeric Aβ/DMSO solution in sterile PBS at 4° C. overnight. Thequality of Aβ preparation is routinely assessed using immunoblotanalysis with the anti-human Aβ monoclonal antibody 6E10 (Signet Lab)that recognizes monomeric and oligomeric forms of Aβ₄₂.

Cannula Infusion technique. Following anesthesia with 20 mg/kg Avertin,mice are implanted with a 26-gauge guide cannula into the dorsal part ofthe hippocampi (coordinates: P=2.46 mm, L=1.50 mm to a depth of 1.30 mm)(Paxinos, G., Mouse Brain in Stereotaxic Coordinates, 2^(nd) Ed. 1998,New York: Academic Press; herein incorporated by reference in itsentirety). The cannulas are fixed to the skull with acrylic dentalcement (made from Paladur powder). After 6-8 days, mice are bilaterallyinfused with oligomeric forms of Aβ₄₂ or vehicle.

Treatment of the APP/PS1 mice: APP/PS1 mice are treated with the testcompounds 2.5 mg/Kg (p.o.) by oral gavage starting from the age of twomonths. The treatment lasts until the behavioral andelectrophysiological tests are completed. Behavioral tests are startedat 3 months of age and last for 3 weeks. Electrophysiological testsstart after the behavioral tests and last about 21 days.

Acute treatment with amyloid-beta: For behavioral testing mice areinfused with amyloid beta through cannulas implanted into dorsalhippocampi. For electrophysiological tests, slices taken from mousehippocampi are perfused with amyloid beta. The test compounds are givenimmediately prior to the behavioral testing or to induction of LTP withthe theta burst.

Behavioral tests include two types of tasks: a) fear conditioning, andb) 2 day radial arm water maze.

Fear conditioning (FC) is assessed as described in Cell 2006, 126, 775and J. Clin. Invest. 2004, 114, 1624 (each herein incorporated byreference in its entirety). First, sensory perception of electric footshock is examined in different groups of mice through the thresholdassessment test. The animals are placed in the conditioning chamber andthe electric current (0.1 mA for 1 sec) is increased at 30 s intervalsfrom 0.1 mA to 0.7 mA. Threshold to flinching (first visible response toshock), jumping (first extreme motor response), and vocalized responseis quantified for each animal by averaging the shock intensity at whicheach animal shows the behavioral response to that type of shock. Nodifferences in the threshold assessment among the different groups ofmice should be found if treatments with the new chemical entity does notaffect animals' sensory threshold. For FC training, mice are placed in aconditioning chamber for 2 min before the onset of a tone (ConditionedStimulus (CS), 30 sec, 85 dB sound at 2800 Hz). In the last 2 sec of theCS, mice are given a 2 sec, 0.7 mA mild foot shock (UnconditionedStimulus, (US)) through the bars of the floor. After the US, the miceare left in the chamber for another 30 s. Freezing behavior, defined asthe absence of movements except for that needed for breathing, is scoredusing Freezeview software (Med Associates, St. Albans, Vt.). Contextualfear learning, a type of memory for which hippocampal function isindispensable, is evaluated 24 hrs after training by measuring freezingresponses for 5 min in the same chamber where the mice have beentrained. Cued fear learning, a type of memory that depends on amygdalafunction, is evaluated 24 hrs after contextual testing. The mice areplaced in a novel context for 2 min (pre-CS test), after which they aregiven a CS for 3 min (CS test), and freezing behavior is measured duringthe first 30 sec that mimic the CS-US conditioning and the remaining 2.5min. In addition, the open-field test is conducted to evaluateexploratory behavior in different groups of mice (Neuroscience 2007,147, 28; J. Neurosci. 2008, 28, 14537 (each herein incorporated byreference in its entirety). The open field consists of a squared arena(72×72×33 cm) made of white acrylic. A 36×36 cm area in the center isdefined as the ‘central compartment’. At the beginning of the test, miceare placed in the center of the open field arena and allowed to movefreely for 1 hour. Amount of time spent in the center compartment vs.the periphery and number of entries into the center compartment isscored. After 24 hours mice are retested for an additional hour.

Radial Arm water maze (RAWM). It has been established that this task isaltered both in Aβ-infused mice and in APP/PS1 animals (see FIG. 7A-B).Reference memory was studied with the 2-day RAWM as described in Nat.Protoc. 2006, 1, 1671 (herein incorporated by reference in itsentirety). For these experiments, visible platform testing was conductedto exclude that visual, motor and motivation deficits affect the mouseperformance (Ann. Neurol. 2004, 55, 801; herein incorporated byreference in its entirety). The task is a hybrid of the Morris WaterMaze (MWM) and the radial arm land maze. The motivation for the animalsis the immersion in water. The mouse needs to swim in 6 alleys (arms)radiating from a central area until it finds a hidden (submerged)platform at the end of one of the arms, based on visual cues placed inthe room. In the old RAWM version of the task, the goal arm varies fromday to day, requiring 21 days of training in wild-type mice to reach thelearning criterion. In the new version of the task, the goal arm is keptconstant for all trials, with a different start arm on successivetrials, such that the learning criterion is reached in 2 days The firstday of the protocol is a training day. Mice are trained to identify theplatform location by alternating between a visible and a hidden platformin a goal arm. The final 3 trials on day 1 and all 15 trials on day 2use a hidden escape platform to force mice to use spatial cues toidentify the location of the goal arm. To avoid learning limitationsimposed by exhausting practice and to avoid fatigue that may result fromconsecutive trials, spaced practice training is established by runningthe mice in cohorts of 4 and alternating different cohorts through the15 training trials over 3 hours testing periods each day. On day 1, avisible platform is placed in a goal location. Mouse 1 of cohort 1 isgently placed in the pool near the perimeter of the wall of the firststart arm (specified on a score sheet) and facing the center of thepool. The number of incorrect arm entries (entries in arms with noplatform) is counted. If the animal enters the incorrect arm it isgently pulled back to the start arm. Each trial lasts up to 1 min.Failure to select an arm after 15 sec is counted as an error and themouse is returned to the start arm. After 1 min, if the platform has notbeen located, the mouse is guided gently through the water by placing ahand behind it to direct it towards the platform. The mouse rests on theplatform for 15 sec. After completing the trial, the mouse is removedfrom the pool, gently towel dried and placed back into its cage under aheat lamp. The goal platform location is different for each mouse. Afterall the mice in the first cohort have had a trial to locate a visibleplatform, the platform is switched from visible to hidden. After eachmouse from cohort 1 completes six alternating trials between visible andhidden platforms, the mice are left to rest under a heating source, andmice from the second cohort are tested in the same way. After completingthe six alternating trials, mice from cohort 2 are returned to theircages to rest. Next, mice from the first cohort complete trials 7-12again using the alternating visible-hidden platform location. Duringresting time for mice from the first cohort, mice from the second cohortcomplete trials 7-12. At this point, all mice will have performed 3hidden platform trials. On day 2, the same procedure is repeated as onday 1 for all 15 trials using only the hidden platform. For dataanalysis, averages for each mouse are calculated using blocks of 3trials.

Electrophysiological tests include a study of long-term potentiation(LTP). Transverse hippocampal slices (400 mm) are cut with a tissuechopper (EMS, PA) and maintained in an interface chamber at 29° C. for90 min prior to recording, as described in Proc. Natl. Acad. Sci. U.S.A.2002, 99, 13217, herein incorporated by reference in its entirety. Theextracellular bath solution consists of 124.0 mM NaCl, 4.4 mM KCl, 1.0mM Na₂HPO₄, 25.0 mM NaHCO₃, 2.0 mM CaCl₂, 2.0 mM MgSO₄, and 10.0 mMglucose, continuously aerated with 95% O₂/5% CO₂ to a final pH of 7.4.Field extracellular postsynaptic responses (fEPSPs) are recorded byplacing the stimulating and recording electrodes in CA1 stratumradiatum. A bipolar tungsten electrode (FHC, Bowdoin, Me.) is used as astimulating electrode, and a glass pipette filled with bath solution isused as a recording electrode. BST is first assessed by plotting thestimulus voltages (V) against slopes of fEPSP, or by plotting the peakamplitude of the fiber volley response against the slope of fEPSP togenerate input-output relations. For LTP experiments, a 15 min baselineis first recorded every minute at an intensity that evokes a response atapproximately 35% of the maximum evoked response. LTP is induced using atheta-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at5 Hz, and each tetanus consisting of 3 ten-burst trains separated by 15sec). Responses are measured as fEPSP slopes expressed as percentage ofbaseline.

Example 16: Efficacy Tests for Cognitive Deficit Attenuation in AβInfused Mouse Models

Animals. Three- to four-month-old C57BL/6J male mice derived from acolony which was bred in our animal facility. They were maintained on a12 h light/dark cycle in temperature- and humidity-controlled rooms.Animals were killed by cervical dislocation followed by decapitation.

Aβ preparation. Aβ42 was prepared as previously described in J. Neurosci2005, 25, 6887 (herein incorporated by reference in its entirety),starting from lyophilized peptide (American Peptide) which was suspendedin 100% 1,1,1,3,3,3-hexafluoro-2-propanol (Sigma-Aldrich) and allowed toevaporate. The resulting clear peptide film was stored at 20° C.Twenty-four hours prior to its use, the film was added to DMSO(Sigma-Aldrich) and sonicated for 10 min. This preparation was dilutedinto the bath solution, vortexed for 30 sec, and incubated at 4° C. for24 hrs. Western blot analysis was routinely utilized to check thebiochemistry of this aged synthetic Aβ as previously described in J.Neurosci 2005, 25, 6887 (herein incorporated by reference in itsentirety).

Electrophysiological studies. Brain slices (400 μm) were cut andmaintained in an interface chamber at 29° C. for 90 min prior torecording, as previously described in J. Neurosci 2005, 25, 6887 (hereinincorporated by reference in its entirety). CA3-CA1 responses wererecorded by means of a stimulating electrode, a bipolar tungstenelectrode, placed at the level of the Schaeffer collateral fibers, and arecording electrode, a glass electrode filled with bath solution.Following assessment of basal synaptic transmission by plotting thestimulus voltages against slopes of field EPSP, a 15 min baseline wasrecorded every minute at an intensity that evokes a response ˜35% of themaximum evoked response. Then, LTP was evoked through a θ-burststimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz andeach tetanus including three 10-burst trains separated by 15 sec).

Behavioral Studies. A) Reference memory was studied with the 2-day RAWMas described in Nat. Protoc. 2006, 1, 1671 (herein incorporated byreference in its entirety). For these experiments, visible platformtesting was conducted to exclude that visual, motor and motivationdeficits affect the mouse performance (Ann. Neurol. 2004, 55, 801;herein incorporated by reference in its entirety). B) Fear Conditioningto examine both contextual and cued learning were assessed, aspreviously described (J. Clin. Invest. 2004, 114, 1624; hereinincorporated by reference in its entirety). For these experiments,threshold assessment test was performed to check sensory perception ofelectric shock in different groups of mice, as previously described (J.Clin. Invest. 2004, 114, 1624; herein incorporated by reference in itsentirety). In addition, the open-field test was conducted to evaluateexploratory behavior, as previously described (J. Neurosci. 2008, 28,14537; herein incorporated by reference in its entirety).

Statistics. Experiments were performed in blind (results expressed asSEM). Significance of differences between 2 groups was determined byt-test for pairwise comparisons or a 2-way ANOVA with repeated measuresfor multiple comparisons. Significance was set at p<0.05.

In a series of experiments, LTP was induced in the presence ofoligomeric Aβ₄₂, a peptide that accumulates in the brains of ADpatients. In these experiments, 200 nM Aβ₄₂ or vehicle were perfusedthrough the bath solution for 20 min prior to application of theθ-burst. As previously demonstrated, Aβ reduced LTP (FIG. 8A)(Neuroreport 1997, 8, 3213; Eur. J. Pharmacol. 1999, 382, 167; Nature2002, 416, 535; Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13217; J.Neurosci. 2005, 25, 6887; Nat. Med. 2005, 11, 556; Proc. Natl. Acad.Sci. U.S.A. 1998, 95, 6448; each herein incorporated by reference in itsentirety). However, MW-181 (1; 10 μM, for 20 min prior to the θ-burst)ameliorated the electrophysiological deficit (FIG. 8A). The relevance toAD pathology of these results was validated by assessing both referencememory and contextual fear memory, two types of memory that are affectedin AD patients. Aβ₄₂ (200 nM, in a final volume of 1 μl over 1 min) orvehicle were bilaterally infused 20 min prior to the 1st trial [for the1st group of tests in a 2-day radial arm water maze (RAWM) testassessing reference memory (Nat. Protoc. 2006, 1, 1671; hereinincorporated by reference in its entirety) and 20 min prior to the 7thtrial (for the 2nd group of tests of the RAWM), into dorsal hippocampusof the animal that had been pre-implanted with a cannula the week beforeor 20 min prior to the foot shock. Aβ reduced the two types of memory(FIGS. 8B and 8C). However, MW-181 (1; 5 mg/Kg, i.p., 30 min before the1st and 2nd group of tests for the RAWM or before training for fearconditioning) rescued the memory defects (FIGS. 8B and 8C). The effectsof the compounds were really due to changes in hippocampal memorymechanisms because in control experiments including visible platformtest, open field, sensory threshold assessment and cued learning, nodifference across groups was found (FIGS. 8D-8I).

Efficacy studies were performed using a second p38α MAPK inhibitor,MW-108 (2), that is more selective than MW-181(1) as it does not hit thep38β isoform or atypical MAPK, CKs and is negative in the Milliporelarge scale kinase and GPCR screens. The compound was used at aconcentration of 10 μM (bath perfusion) for the LTP experiments and 5mg/Kg (i.p.) for the behavioral experiments using the same experimentalparadigms as for MW-181 (1). These experiments showed that MW-108 (2)was still capable of rescuing the LTP deficits, as well as the defectsin contextual fear memory and reference memory due to exogenous Aβ₄₂application (FIG. 9A-I).

LTP was induced in the presence of oligomeric Aβ₄₂. As expected fromprevious studies, Aβ reduced LTP (FIG. 9A) (J. Clin. Invest. 2004, 114,1624; J. Neurosci. 2005, 25, 6887; Ann. Neurol. 2004, 55, 801; J.Neurobiol. Aging 2009, 30, 257; Eur. J. Med. Chem. 2013, 60, 348; eachherein incorporated by reference in its entirety). Importantly, MW-108(2) ameliorated the Aβ-induced electrophysiological deficit (FIG. 9A),indicative of rescue of synaptic plasticity impairment.

The initial results were extended by testing whether MW-108 (2) couldattenuate cognitive deficits in two AD-relevant behavioral tasks thatmeasure two types of memory dysfunction: the radial arm water maze(RAWM) to measure reference memory (Eur. J. Med. Chem. 2013, 60, 348;herein incorporated by reference in its entirety) and fear conditioningto measure contextual fear memory (J. Clin. Invest. 2004, 114, 1624;herein incorporated by reference in its entirety). Aβ₄₂ or vehicle wasbilaterally infused into the dorsal hippocampus of mice. Aβ-infused miceshowed memory deficits, exhibiting more performance errors compared tovehicle-infused mice (FIG. 9B). Treatment with MW-108 (2) rescued thememory defects (FIG. 9B). In the fear conditioning task, Aβ₄₂ caused acontextual fear memory deficit which was prevented by MW-108 (2)administration (FIG. 9C). Control experiments, including visibleplatform test, open field, sensory threshold assessment and cuedlearning, showed no differences across groups (FIG. 9D). These datadocument that MW-108 (2) attenuates AD-relevant impairments in synapticplasticity and memory.

Example 17: Efficacy Tests in Transgenic Mouse Models

Experiments were also validated in an Aβ depositing mouse model, theAPP/PS1 mouse (Nat. Med. 1998, 4, 97, herein incorporated by referencein its entirety). These mice are well characterized with respect to ADpathology, and begin to show synaptic and memory impairment as early as3 months of age (Ann. Neurol. 2004, 55, 801; Nat. Med. 1998, 4, 97; Exp.Neurol. 2001, 171, 59; Neurobiol. Dis. 1999, 6, 231; and Neurochem. Res.2003, 28, 1009; each herein incorporated by reference in its entirety).In these experiments, (1) (MW-181) was given daily, p.o. (2.5 mg/kg)from the age of 2 months until 3-4 months of age.

As shown in FIG. 10, compound 1 (MW-181) ameliorated theelectrophysiological deficits without affecting WT mice (FIG. 10A).Additionally, WT mice exhibited ˜1 error at the end of the second daywith the RAWM task. APP/PS1 in turn failed to learn and made ˜3 errors.Treatment with compound 1 (MW-181) reduced the memory deficit in doubleTgs without affecting memory in WT mice (FIG. 10B). Similar results wereobserved when double Tgs mice performed FC to assess contextual fearmemory (FIG. 10C). Regarding the behavioral experiments, controlexperiments were performed including visible platform test, open fieldand sensory threshold assessment which did not reveal any differenceacross groups.

As shown in FIG. 11, MW-108 (compound 2) was also tested in the APP/PS1mouse model and rescues deficits in LTP, fear memory and referencememory in amyloid depositing mice.

Representative compounds MW-077 (7), MW-125 (9) and MW-150 (27) weretested to rescue defects of synaptic plasticity and memory in APP/PS1mice. Daily p.o. treatment starting from 8 weeks of age until the end ofthe experiment at 3-4 months of age, at a concentration of 2.5 mg/kg,recued impairment of LTP and memory, including contextual fear and shortterm reference memory. The beneficial effects of the compounds regardingLTP and memory were due to an effect on the mechanism underlying thedefects in these phenomena and not to an additive effect on them becauseadministration of the drug to WT littermates did not change the amountsof LTP and behavioral performance. Controls were performed using cuedconditioning the check for impairment of amygdala function, thresholdassessment test to check sensory perception of electric shock indifferent groups of mice, visible platform testing to exclude thatvisual, motor and motivation deficits affect performance, and theopen-field test to evaluate exploratory behavior. None of these testsshowed an effect of the compounds, indicating that the effect is due totrue action on the cognitive mechanisms.

As shown in FIG. 12A-C, MW-077 (7), MW-125 (9) and MW-150 (27) rescuethe defect in contextual fear learning in APP/PS1 mice. The compoundsdid not affect the performance in WT littermates. Mice were treated (2.5mg/kg, daily, oral gavage) with the compound from the age of 2 monthsuntil behavioral testing that occurred at ˜3 months of age.

As shown in FIG. 13A-C, MW-077 (7), MW-125 (9) and MW-150 (27) (2.5mg/kg, daily, oral gavage) do not affect cued fear memory in APP/PS1mice.

As shown in FIG. 14-19, MW-077 (7), MW-125 (9) and MW-150 (27) (2.5mg/kg, daily, oral gavage) did not animal performance with open field(FIGS. 14-16) and sensory threshold (FIGS. 17-19), meaning that thecompounds do not affect anxiety status of the animal and its ability tofeel the noxious stimulus.

As shown in FIGS. 20-21, controls of visible platform testing show thatMW-077 (7), MW-125 (9) and MW-150 (27) (2.5 mg/kg, daily, oral gavage)do not affect performance with visible platform tasks in APP/PS1 mice.

As shown in FIG. 22, MW-077 (7), MW-125 (9) and MW-150 (27) rescuedefects in spatial memory in APP/PS1 mice. The compounds did not affectperformance in WT littermates. Mice were treated with the compounds (2.5mg/kg, daily, oral gavage) from the age of 2 months until behavioraltesting that occurred at ˜3 months of age.

As shown in FIG. 23, MW-077 (7), MW-125 (9) and MW-150 (27) rescuedefects in LTP in APP/PS1 mice. The compounds did not affect LTP in WTlittermates. Mice were treated with the compounds (2.5 mg/kg, daily,oral gavage) from the age of 2 months until the sacrifice of the animalswhich occurred between 3-4 months.

Methods to screen compounds for treatment of Alzheimer's Disease arealso described, for example, in WO 11/072243 and WO12/088420, eachherein incorporated by reference in its entirety.

Example 18: Amyotrophic Lateral Sclerosis (ALS)

Animal models: SOD1 transgenic mice are used as a model for ALS. Thesemice have the mutant human SOD1 (G93A) substitution. Motor impairmentstarts at 12 weeks of age and includes changes in body weight, reductionin grip strength and general motor activity, which can be assessed usingthe tests outlined below. This phenotype continues to progress untildeath at about 19 weeks.

Body Weight: The body weight of the mice is recorded at regularintervals throughout the duration of the study. SOD1 (G93A) mice exhibita reduction in bodyweight starting at 12 weeks of age in males and 16weeks of age in females. Body weight loss continues to progress withdisease progression.

Grip Strength: Grip strength is used to assess muscular strength in limbmuscles. The mouse is lowered toward the platform and gently pulledbackwards with consistent force by the experimenter until it releasesits grip. The grip force is recorded through a strain gauge. SOD1 (G93A)mice exhibit a decline in forelimb grip strength, from about 9 weeks ofage, and continuing to decline as the disease progresses. SOD1 (G93A)mice also show a decline in hind limb grip strength, starting at about 9weeks of age, and continuing to decline with disease progression.Finally, SOD1 (G93A) mice exhibit a decline in hind limb grip strength,starting from around 9 weeks of age, and continuing to decline withdisease progression.

Rotarod: Motor coordination and exercise capacity are assessed throughthe rotarod. Tests are performed on separate days, with multiple trialsper day. Mice are placed on the rotarod and the speed of the rotation ofthe rod is gradually and uniformly increased over a maximum period oftime. The time that each mouse remains on the rotating rod beforefalling is recorded. SOD1 (G93A) mice demonstrate a decline in motorfunction from 12 weeks of age.

Survival: Survival of the mice is recorded and can be cumulativelyfollowed as the population in the various treatment groups decline. SOD1(G93A) mice begin to show decreased survival at about 120 days, with anincreasingly steep decline and zero survival at about 150 days.

Treatment with the test compound starts from the age of two months. Thetreatment lasts until the death of the animal.

Example 19: Parkinson's Disease (PD)

Animal model: The MPTP mouse model of Parkinson's disease is thought tomimic more closely the behavioral pathology of Parkinson's disease,compared to other models. Mice exhibit Parkinson's-like symptomsfollowing systemic injection of the pyridine toxin MPTP, which producesa loss of striatal dopamine (DA) nerve terminal markers and, at higherdoses, death of DA neurons in the substantia nigra. The process ofterminal loss and degeneration takes 6-9 days following MPTP injection.

Treatment: The test compounds can be administered both before the MPTPis given (to evaluate neuroprotection), and/or while the toxin is activein the first week post-injection (to evaluate preservation of dopaminefunction).

Neurochemical assay: Tyrosine Hydroxylase (TH) Immunohistochemistry(Quantitative Morphology) and Dopamine turnover. Determination of DA,DOPAC and HVA concentrations in the striata of control and MPTP-lesionedmice. The DA and DOPAC levels are significantly reduced, inMPTP-lesioned mice as compared to control groups.

Behavior Testing: In mice, MPTP injections produce a temporary increasein hyperactivity, a long-lasting increase in wall-supported rearing inthe open field enclosure (relative to “free” rearing in the center), alasting increase in foot slippage (foot faults in a grid-walking test)and a lasting impairment in treadmill activity (using several measuresof gait). The hyperactivity returns to baseline after 2 weeks but theother three measures of impaired balance remain abnormal indefinitely.Improvements in the latter measures following drug treatment correlatewith increased dopamine neuron presence (increased numbers ofTH-positive cells) in the substantia nigra and increased dopamineactivity in the striatum.

Methods to screen compounds for treatment of Parkinson's Disease arealso described, for example, in WO 11/072243 and WO12/088420, eachherein incorporated by reference in its entirety.

Example 20: Huntington's Disease (HD)

Animal model. Several mouse models of HD have been engineered. Out ofall of them, YAC128 is advantageous because a) it expresses the fulllength protein, and thus Lysine 444; b) it has a faster age of onsetthan knock-in model; c) the aggregation pattern is more reminiscent toadult onset HD; and d) reports of some neuronal loss as measured by darkcell degeneration in the striatum. This model will therefore permitsimultaneous testing of the importance of transcriptional dysregulationin HD as well as the importance of selective degradation of mutanthuntingtin protein.

Treatment: The test compound is administered by gavage daily to 8 weekold mice up to 10 months. The primary endpoints of therapeuticeffectiveness are evaluated by testing monthly motor behaviors includingrotarod, gait analyses and open field, starting at 10 weeks of age asdescribed in Cell 2000, 101, 57, herein incorporated by reference in itsentirety, to determine if the new compound administration leads to adelay in phenotype onset. To determine if the new compoundadministration leads to a decrease of mutant htt protein levels, a smallcohort of (n=3) mice is sacrificed after 1 month and analyzed forSDS-soluble and insoluble mutant htt protein using EM48 (Chemicon) and3B5H10 (Sigma). EM48-positive intracellular inclusions, stereologicalcounts of striatal neurons (NeuN-positive), dark cell degeneration andbrain weights are collected starting at 12 months of age. Part of thebrains is utilized for measuring histone acetylation levels. Forbehavioral experiments mice are divided into different cohorts of 12mice per group (see group distribution below). Although the studycontinues up to the age of 12 months, within 4 months of the study onewill learn whether chronic administration of the new compound has apositive input in mutant huntingtin protein levels, as well as whetherthe new compound can delay the age of phenotypic onset.

The phenotypic analysis of these mice includes: Rotarod, OpenFieldmeasurement of SDS-soluble and insoluble mutant htt protein,EM48-positive intracellular inclusions, stereological counts of striatalneurons (NeuN-positive) and brain weights.

Accelerating Rotarod: This assay is a common assay used to monitor motorcoordination in mouse models of Huntington's disease (HD). After atraining period to acclimatize the mice to the apparatus, mice areplaced on a rod rotating at 5 rpm. Over the test period of 300 sec, therod increases its rotational speed from 5 rpm to 40 rpm. The time atwhich the mouse falls off of the rod is recorded. Each mouse is subjectto 3 runs per day (shown as a per day average) across 3 days (d1, d2,d3). Typically 8 to 12 mice per group are used in this assay.

Open field: This assay is a common assay used to monitor totalspontaneous locomotor activity and exploratory behavior in mouse modelsof Huntington's disease (HD). The spontaneous locomotor activity of theanimals is measured using an automated photo-beam open-field system(Med-Associates, St Albans, Vt.). Mice are placed individually in thecenter of a clear open-field chamber (27.9 cm×27.9 cm×20 cm), and theirhorizontal and vertical activities are measured immediately for 30 minwith three 16-beam FR arrays. Locomotor activity is assessed as thedistance traveled in 5-min intervals.

Methods to screen compounds for treatment of Huntington's Disease arealso described, for example, in WO 11/072243 and WO 12/088420, eachherein incorporated by reference in its entirety.

Example 21: Oxygen Glucose Deprivation

Slice preparation. Animals were anesthetized using urethane (20%solution, 1 mL/100 g body weight) via intraperitoneal injection and thendecapitated after disappearance of the tail pinch reflex. After brainremoval, horizontal sections (400 μm in thickness), containing theentorhinal area, were made on a Vibratome. All steps were performed inice-cold oxygenated ACSF solution. Before recording, slices were storedfor at least 1 h in a recovery chamber containing oxygenated ACSFsolution at room temperature. For electrophysiology, slices wereperfused at a rate of 2.5-3 mL/min with oxygenated ACSF at 33±1° C.Extracellular field potentials (FPs) were evoked in layer II/III in 3response to electrical stimulation of layer II (Origlia et al., Receptorfor advanced glycation end product-dependent activation of p38mitogen-activated protein kinase contributes to amyloid-β-mediatedcortical synaptic dysfunction. J Neurosci 2008, 28:3521-3530; Origlia etal., Microglial Receptor for Advanced Glycation End Product-DependentSignal Pathway Drives β-Amyloid-Induced Synaptic Depression andLong-Term Depression Impairment in Entorhinal Cortex, Journal Neurosci2010, 30(34): 11414-11425; each herein incorporated by reference in itsentirety). The amplitude of the FPs was used as a measure of the evokedpopulation excitatory current. All FPs had peak latency between 4.5 and6 ms. Baseline responses were obtained with a stimulation intensity thatyielded 50-60% of maximal amplitude. After 15 min of stable baselinerecordings slices were perfused for 10 minutes with deoxygenatedglucose-free ACSF (glucose was substituted with D-mannitol at equimolarconcentration), to obtain a transient oxygen/glucose deprivation (OGD).The amplitude of FP was monitored every 20 s and averaged every threeresponses by online data acquisition software. Effects on synapticfunction were evaluated either during ischemia (as the average of FPsamplitude during the last 3 min of 10 min OGD application) or as therecovery of FPs calculated as the averaged relative amplitude of FPsrespect to the baseline values after reintroduction of regular ACSF(from 41st to 50th min after the end of OGD). Synaptic depressioninduced by OGD is ameliorated by MW-150 (FIG. 24). Slices werecontinuously perfused with ACSF containing 10 μM of the compound.

Example 22

Dose response curve for MW-150 during assessment of associative andspatial memory deficit in APP/PS1 transgenic mice. Mice wereadministered orally either saline vehicle or different concentrations ofMW-150 daily from 8 weeks of age until 3-4 months when associative andspatial memories were assessed through contextual fear memory (FIG. 25A)and RAWM (FIG. 25B). RAWM errors correspond to the number of errors thatmice (3-15 per test) made at the last set of trials.

Example 23

Results in two distinct pathology progression models demonstrate theability bring about pharmacological efficacy with MW-150 repeat dosing.Further, the results in a battery of behavioral tests are consistentwith a true hippocampus-dependent mechanism of action in attenuation ofmemory deficits. Repeat dosing with MW-150 did not bring about anyobserved adverse events within the targeted physiological axis orcontrol behaviors in aged or diseased animals. The selective improvementin cognitive behavior was also brought about with no effect on amyloidplaque load (Wilcock, D. M., and Colton, C. A. (2009) Immunotherapy,vascular pathology and microhemorrhages in transgenic mice. CNS Neurol.Disord.: Drug Targets 8, 50-64, herein incorporated by reference in itsentirety).

Histopathology. Immunohistochemistry was performed using the VentanaBenchMark Ultra automated platform. Tissue sections were firstdeparaffinized using Ventana's “ez-prep” solution. Antigen retrieval wasperformed by treatment at 95° C. for 54 minutes using Ventana's CC1 (pH7.3) solution, followed by treatment with 0.3% hydrogen peroxide toblock endogenous peroxidase. Tissue sections were then incubated inprotein-free block (Biocare's background sniper) for 15 min to inhibitnonspecific binding. Primary antibody (anti-Aβ 6E10 at 1:400, BiocareMedical) was incubated for 32 min at room temperature. Detection wasperformed using Ventana's ultraview DAB kit, and counterstaining withGill hematoxylin solution. A board-certified neuropathologist, who wasblinded to the treatment versus the control group, analyzed a sagittalsection from each mouse and counted the total number of well-formedβ-amyloid plaques.

MW-150 treatment improves cognitive performance in the absence of effecton Aβ plaque burden. Sections of cortex from APP/PS1 transgenic micetreated with vehicle (FIG. 26A) or MW-150 (FIG. 26B) were stained with6E10 anti-Aβ antibody (10× objective; 100× magnification). (FIG. 26C)Quantification by a board-certified neuropathologist blinded to thetreatment groups was done by analysis of a brain sagittal section fromeach mouse in which the total number of well-formed β-amyloid plaques inthe entire section were counted. Error bars show standard error of themean (n=4 for each group). (FIG. 26D) In a separate experiment withAPP/PS1 knock-in (KI) mice, Aβ plaques were quantified from KI micetreated with MW-150 or vehicle. In both AD mouse S15 models, there areno effects of MW-150 treatment on the amyloid plaque burden.

Although the invention has been described and illustrated in theforegoing illustrative embodiments, it is understood that the presentdisclosure has been made only by way of example, and that numerouschanges in the details of implementation of the invention can be madewithout departing from the spirit and scope of the invention, which islimited only by the claims that follow. Features of the disclosedembodiments can be combined and/or rearranged in various ways within thescope and spirit of the invention to produce further embodiments thatare also within the scope of the invention. Those skilled in the artwill recognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific embodimentsdescribed specifically in this disclosure. Such equivalents are intendedto be encompassed in the scope of the following claims.

The invention claimed is:
 1. A method of increasing long-term potentiation in a subject having a neurodegenerative disease comprising administration of a therapeutically effective amount of a compound of formula (I), or a composition comprising a compound of formula (I),

wherein X₁ is N and X₂ is CH, or X₁ is CH and X₂ is N; R¹ is —N(R⁴)₂, cyclopropyl, or R⁵-piperidin-4-yl; R² is independently D or halogen; R³ is naphthyl, quinolinyl, or isoquinolinyl, wherein said naphthyl, quinolinyl, or isoquinolinyl is optionally independently substituted with at least one D, halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D; R⁴ is independently H, (C₁-C₄)-alkyl, (C₁-C₄)-alkyl substituted with at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the nitrogen to which they are attached form a 3-7 membered heterocyclic ring, wherein one of the carbon atoms is optionally replaced with NR⁶, O or S, and wherein the 3-7 membered heterocyclic ring is optionally substituted with a (C₁-C₃)-alkyl; R⁵ is H or (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least one D; R⁶ is H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with at least one D, or pyrimidin-2-yl; and n is an integer from 0-4; or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein R⁴ is independently H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the nitrogen to which they are attached form a 3-7 membered heterocyclic ring, wherein one of the carbon atoms is optionally replaced with NR⁶, O or S; and R⁶ is H, (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least one D.
 3. The method of claim 2, wherein R³ is naphthyl, quinolinyl, or isoquinolinyl, wherein said naphthyl, quinolinyl, or isoquinolinyl is optionally independently substituted with at least one D, halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D.
 4. The method of claim 1, wherein the compound is selected from


5. The method of claim 1, wherein the compound is


6. A method of improving memory in a subject having a neurodegenerative disease comprising administration of a therapeutically effective amount of a compound of formula (I), or a composition comprising a compound of formula (I),

wherein X₁ is N and X₂ is CH, or X₁ is CH and X₂ is N; R¹ is —N(R⁴)₂, cyclopropyl, or R⁵-piperidin-4-yl; R² is independently D or halogen; R³ is naphthyl, quinolinyl, or isoquinolinyl, wherein said naphthyl, quinolinyl, or isoquinolinyl is optionally independently substituted with at least one D, halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D; R⁴ is independently H, (C₁-C₄)-alkyl, (C₁-C₄)-alkyl substituted with at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the nitrogen to which they are attached form a 3-7 membered heterocyclic ring, wherein one of the carbon atoms is optionally replaced with NR⁶, O or S, and wherein the 3-7 membered heterocyclic ring is optionally substituted with a (C₁-C₃)-alkyl; R⁵ is H or (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least one D; R⁶ is H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with at least one D, or pyrimidin-2-yl; and n is an integer from 0-4; or a pharmaceutically acceptable salt thereof.
 7. The method of claim 6, wherein R⁴ is independently H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the nitrogen to which they are attached form a 3-7 membered heterocyclic ring, wherein one of the carbon atoms is optionally replaced with NR⁶, O or S; and R⁶ is H, (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least one D.
 8. The method of claim 7, wherein R³ is naphthyl, quinolinyl, or isoquinolinyl, wherein said naphthyl, quinolinyl, or isoquinolinyl is optionally independently substituted with at least one D, halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D.
 9. The method of claim 6, wherein the compound is selected from


10. The method of claim 6, wherein the compound is


11. A method of treating ischemia in a subject in need thereof comprising administration of a therapeutically effective amount of a compound of formula (I), or a composition comprising a compound of formula (I),

wherein X₁ is N and X₂ is CH, or X₁ is CH and X₂ is N; R¹ is —N(R⁴)₂, cyclopropyl, or R⁵-piperidin-4-yl; R² is independently D or halogen; R³ is naphthyl, quinolinyl, or isoquinolinyl, wherein said naphthyl, quinolinyl, or isoquinolinyl is optionally independently substituted with at least one D, halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D; R⁴ is independently H, (C₁-C₄)-alkyl, (C₁-C₄)-alkyl substituted with at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the nitrogen to which they are attached form a 3-7 membered heterocyclic ring, wherein one of the carbon atoms is optionally replaced with NR⁶, O or S, and wherein the 3-7 membered heterocyclic ring is optionally substituted with a (C₁-C₃)-alkyl; R⁵ is H or (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least one D; R⁶ is H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with at least one D, or pyrimidin-2-yl; and n is an integer from 0-4; or a pharmaceutically acceptable salt thereof.
 12. The method of claim 11, wherein R⁴ is independently H, (C₁-C₃)-alkyl, (C₁-C₃)-alkyl substituted with at least one D, (C₃-C₅)-cycloalkyl, or each R⁴ together with the nitrogen to which they are attached form a 3-7 membered heterocyclic ring, wherein one of the carbon atoms is optionally replaced with NR⁶, O or S; and R⁶ is H, (C₁-C₃)-alkyl, or (C₁-C₃)-alkyl substituted with at least one D.
 13. The method of claim 12, wherein R³ is naphthyl, quinolinyl, or isoquinolinyl, wherein said naphthyl, quinolinyl, or isoquinolinyl is optionally independently substituted with at least one D, halogen, (C₁-C₃)-alkoxy, or (C₁-C₃)-alkoxy substituted with at least one D.
 14. The method of claim 11, wherein the compound is selected from


15. The method of claim 11, wherein the compound is 